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The Diastatic Enzymes of Wheat 

Flour and Their Relation to 

Flour Strength 



A THESIS SUBMITTED TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

LOUYS A. RUMSEY, B.S., M. S. 

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR 
THE DEGREE OF DOCTOR OF PHILOSOPHY 

June, 1922 



Chicago^ III. 



The Diastatic Enzymes of Wheat 

Flour and Their Relation to 

Flour Strength 



A TH1-:SIS SiniMlT'l'l'D TO THE FACULTY OF THE GRADUATE 
SCHOOL OF THE UNIVERSITY OF MINNESOTA 

BY 

LOUVS A. RUMSEY, B.S.., M. S. 

L\ I'ARTLVL FULFILLMENT OF THE REQUIREMEXTS FOR 
THE DEGREE OF DOCTOR OF I'HILOSOFHY 

June, 1922 



Chicago, III. 






LIBRARY OF CONGRFSS i 

RECEIVED j 

DOOUMliNTS DiViolOi. 



Research Fellowship Plan Under Which Fellows of the American 

Institute of Baking Have Been Enrolled as Graduate 

Students of the University of Minnesota. 

The American Institute of Baking- in the fall of 1920 detailed two 
research fellows in the chemistry of baking- to work on suitalile prob- 
lems as graduate students of the University of Minnesota. These 
fellows were regularly registered in the Graduate School of the Uni- 
versity. They pursued such courses as ordinarily constitute a study 
program of candidates for the doctorate in philosophy, majoring in 
the Division of Agricultural Biochesistry. Research problems were 
selected and outlined in conference with their advisors in this Division, 
actual work on the problems being pursued, by special agreement, 
chiefly in the laboratories of the American Institute of Baking. Theses 
based on this research were duly presented in partial fulfillment of 
the degree of Doctor of Philosophy, and accepted by committees of 
the Graduate School of the University. These theses are. by agree- 
ment with the graduate faculty of the Division of Agricultural Bio- 
chemistry, published by their respective authors as bulletins of the 
American Institute of Baking. 



TABLE OF CONTENTS 



INTRODUCTION 5 

Factors Studied in Their Relation to Mour Slrenyth 5 

Gluten Content 5 

Protein Analysis 5 

Colloidal Properties 5 

Climate 6 

Electrolytes • 6 

Electrometric Methods • 7 

Gas Retention and Gas Production 7 

Carbohydrate Content 7 

Enzymes • 8 

Biochemical Methods of Study 8 

PURPOSE OF INVESTIGATION 9 

HISTORICAL 10 

Definition of Diastase 10 

Review of Literature on Diastase 10 

Preliminary Discussion -17 

1. Liquifying and Saccharogenic Action 17 

2. Resistance of Different Starches to Diastatic Action 19 

3. Difference Between Autolytic and Extracted Enzyme Activity 20 

EXPERIMENTAL PART 23 

1. The Problem ....Z3 

2. The Materials 24 

3. History and Description of Flour Samples 25 

4. Baking Tests 26 

Formula 26 

Method 27 

Data and Score of Baked Loaves 29 

5. Clarification of Cereal Extracts and Suspensions by Sodium 

Tungstate 30 

Efficiency H 

Effect on Reducing Sugar Determination 34 

Elimination of Filtration Z7 

Inhibition of Enzyme Activity 41 

6. Effect of Concentration of Flour-Water Suspension on Diastatic 

Activity 44 

7. Method for Measuring Diastatic Power in Flours 48 

8. Effect of Temperature on the Activity of Wheat Flour Diastase 50 

9. Effect of Time on the Activity of Wheat Flour Diastase 52 

10. The Effect of pH on the Activity of Wheat Flour Diastase 54 

11. Buffer Action and Buffer Valves 56 

12. The Relative Diastatic Powers of Fourteen Samjiles of Wlieat Fiour.60 

13. Diastatic Activity During Fermentation of the Dough 62 

Method ' 64 

In Normal Doughs (i(\ 

In Doughs with Added Diastatic Enzymes 66 

Discussion of Data ' 66 

THE RELATION OF DIASTATIC POWER TO DIFFERENT FORMS 
OF STARCH . 70 

ANALYTICAL DATA ON FLOUR SAMPLES.. .. 73 

SUMMARY yT, 

BIBLIOGRAPHY 7S 

BIOGRAPHICAL 85 



THE DIASTATIC ENZYMES OF WHEAT FLOUR 

AND THEIR RELATION TO FLOUR STRENGTH 

By Louys A. Rumsey 

INTRODUCTION 

The role which the enzymes play in the production of a good loaf 
of bread and their importance as contributing factors in the strength 
of flour is a matter of real concern to both the baker and the miller. 
After years of chemical and physical investigation of wheats and 
flours the cereal technologist is still confronted with the problem of 
finding some factor, or group of factors, the determination of which 
will furnish a sure index to the baking strength of a given flour. In 
spite of laboratory control in the blending and milling of wheats 
the wide variations in the ability of the corresponding flour to produce 
a "large, well piled loaf" (Humphries and Biffen, 1907.) still neces- 
sitates an actual baking test on each flour. 

Gluten Content. — Because of the unique property of wheat pro- 
teins to form a tenacious, elastic framework of gluten for the reten- 
tion of the aerating gas, attention was first turned to the amount 
and character of the gluten content. The numerous contributions 
of Osborne (1895 to 1907), Guthrie (1896), (1900), Fleurent (1896), 
Snyder (1899), Guess (1900), Humphries (1907-1910). Guthrie and 
Norris (1909), Wood and Hardy (1909), Bailey (1916), Blish (1916), 
Upson and Calvin (1916), Swanson and Tague (1917). Gortner and 
Doherty (1918), and others have led to the conclusion that in the 
proteins of the flour, and especially in the gluten, is to be found the 
most important basis for differences in baking value. 

Protein Analyses. — On the other hand Fleurent (1901), Snyder 
(1901), Shutt (1905-1908), Norton (1906). Chamberlain (1906), 
Wood (1907), Thatcher (1907), Armstrong (1910), Ladd and Bailey 
(1910), Bailey (1913-1914), Blish (1916), and Martin (1920), have 
shown that percentage relationships from protein analyses would not 
sufflce for the predetermination of flour strength. Neither does the 
accurate characterization of the proteins by Osborne and his co- 
workers apply directly to the problem of strength. 

Colloids. — A more promising point of attack appeared to lie in the 
colloidal studies of the wheat gluten, the work on which is well sum- 
marized to 1918 by Gortner and Doherty. Physico-chemical studies 
of the gluten colloids (Gortner and Sharp, 1921.) involving viscosity 
measurements of the wheat flour proteins at dififerent hydrogen ion 
concentrations, being conducted in the Division of Agricultural Bio- 



chemistry, University of Minnesota, give promise of furnishing a most 
valuable index of strength for those flours in which strength is deter- 
mined by gluten quality. 

Climate, — The experience of the millers as well as the direct ex- 
perimental evidence summarized by the reports of LeClerc (1909, 
1910, 1912, 1914), Bailey (1913), and Thatcher (1913), have demon- 
strated that environment is the most important factor in the develop- 
ment of the wheat kernel. The biological differences in the rela- 
tionships of various constituents are most marked when the same 
seed is grown under different climatic conditions. While changes of 
soil impose certain changes in the protein, ash, and carbohydrate con- 
tent of the same wheats, the correlation is by no means as simple 
and direct as that between climate and "strength" of the wheat flour. 
Likewise different varieties and types of wheat respond most readily 
to climatic changes. The corresponding "strength" characteristics 
have consequently come to be regarded as typical of certain grow- 
ing regions. We should expect the same changes to be manifested 
in the respective enzymes of the wheat kernel as carried over into 
the flour. For instance, a hard spring wheat, cut and shocked be- 
fore it is biologically ripe, should be expected to show a different 
diastase activity than a softer wheat grown in the dry and semi-arid 
regions where the "dead ripe" grain is allowed to stand in the field 
under a hot svm and hot winds for days before cutting and thresh- 
ing. Humphries (1910) came to the conclusion that such climatic 
factors did operate so as to change the diastatic activity of the flours 
from these wheats as well as their other strength characteristics. 

Electrolytes. — The addition of various electrolytes as flour "im- 
provers" and as yeast nutrients, bears a close relationship to the prob- 
lem of gluten quality. As early as 1905 Fleurent began to inves- 
tigate the effects of different acids and salts on gluten. Wood (1907), 
Shutt (1908), Wood and Hardy (1909), Humphries (1910), Wil- 
lard and Swanson (1912), and Olson (1917), are among the earlier 
contributors to the study of electrolytes and their relation to strength. 
Numerous experiments with flour "improvers" and yeast stimulants 
have furnished a basis for the widespread use of various electrolytes 
on a commercial scale for the improvement of the baked loaf. The 
most of these investigations are included in the reports of Jessen- 
Hansen (1911), Bacon (1916), Kohman (1915), and (1916)', Koh- 
man, Hoffman, et al (1916), Hoffman (1917), Thomas (1917), Hen- 
derson, Fenn, and Cohn (1919), and Henderson, Cohn, Cathcart, 
Wachman and Fenn (1919). The real action of such added ma- 
terials, for example on the gluten or on the enzymes, is but poorly 
understood as yet, and the whole subject needs further research. 



Electrometric Methods. — The introduction of electrometric meth- 
ods into the field of biological research has both facilitated and stim- 
ulated new methods of attack on the problem of flour strength. De- 
terminations of the hydrogen ion concentration of flour, dough, and 
bread have helped to clear up some vexing questions, the most notable 
of which are : the control of undesirable organisms in bread, Cohn 
and Henderson (1918), Cohn, et al (1918), Morrison and Collatz 
(1921); the optimum acidity for proper fermentation, Jessen-Han- 
sen (1911), Cohn, Cathcart and Henderson (1918) ; the optimum 
hydrogen ion concentration for enzyme actions, Sherman and Wal- 
ker (1917), Sherman, Thomas and Baldwin (1919) ; and the lack of 
relationship between true and titrable acidity, Swanson and Tague 
(1919). Quite recently Bailey (1918), Bailey and Collatz (1921), 
and Bailey and Peterson (1921), have applied electrometric meas- 
urements successfully to the determination of electrolyte content 
of flours in their relation to flour grade. Collatz and Bailey (1921), 
have likewise been able to measure results of phytase activity 
by means of electrical conductivity measurements. 

Gas Retention and Gas Production. — Wood in 1907, Humphries 
in his discussion of Baker and Hulton's paper on flour strength 
1908, and Armstrong in 1910, all emphasized their opinions as 
to the importance of gas retention by a dough in relation to the gas 
production during fermentation. In 1916 Bailey proposed a method 
for comparative measurements of this ratio by means of an "ex- 
pansimeter". Martin (1920) has published the results of compara- 
tive tests on the gas producing capacity, and gas retaining power 
of flours based on the fermentation method of Wood. He summarizes 
these two factors in flour strength by ascribing to a strong flour 
a minimum gas-producing capacity with a high gas-retaining capac- 
ity. Bailey and Weigley (1922) have just completed a report of some 
new studies on the retention of carbon dioxide in doughs in relation 
to gas production which lead to the interesting preposition that part 
of the ripening of the dough, and much of the spring in the oven 
is due to the carbon dioxide gas dissolved in the dough. Also their 
conclusions relative to the ratio of gas production to gas retention 
factors are in agreement with the hypothesis of Humphries, Arm- 
strong, Biffen and others that the carbon dioxide production-rate 
is one of the important factors in flour strength. 

Carbohydrate Content of Flour. — The intimate relationship be- 
tween alcoholic fermentation and the proper aeration of the dough 
necessitated data on the carbohydrate content of wheat flour. But 
the analyses of Teller (1898), (1912), Stone (1896 and 1897), Brown, 
Morris, and Millar (1897), K5nig (1898), Shutt (1907, 1908), Alway 



and Hartzell (1909), Liebig (1909), Jago (1911), Thatcher (1913, 
1915), Olson (1917), all verify the same conclusion, namely, that 
the small amounts of soluble carbohydrates naturally occurring in 
flour are insignificant in comparison to the amounts required for 
proper fermentation in the dough batch. 

It should be borne in mind that there may be some correlation be- 
tween the natural sugar content of the wheat and its relative state 
of biological ripeness. The investigations of Teller, Shutt, Thatcher, 
and Olsen, just referred to, would indicate the effect of climatic factors 
on the sugar content in flour milled from certain wheats. 

Enzymes." — A natural corollary to the study of carbohydrate content 
is the investigation of those enzymes which are responsible for the pro- 
duction of carbohydrates available for yeast growth and fermenta- 
tion, and those which alter and "condition" the gluten. Wood (1907), 
and Humphries (1907, 1910), Liebig (1909), recognized the greater 
importance of diastatic action than of sugar content in panary fer- 
mentation, and they reported measurements of the activity of such 
enzymes. These earlier experiments, along with other preliminary 
work on the enzymes of wheat flour bore evidence of their impor- 
tance as contributory factors in the production of a good loaf. In addi- 
tion to diastase, the other enzymes which have received the most 
study relative to their action in flour and dough are the proteases, 
the phytase, and catalase. The presence of a cytase, acting upon 
the outer surface or envelope of starch cellulose, has been postulated, 
but the evidence for the activity of this enzyme in flour is incon- 
clusive. For the purpose of this report the activities of these dif- 
ferent enzymes can best be considered along with the discussion 
of diastase. 

Strength Probably Due to the Interaction of Several Factors. — It 
has become evident from the accumulated data resulting from 
years of investigation that no single analytical factor has yet been 
found which will suffice to predetermine the baking strength of a 
given flour. The baking test cannot as yet be replaced by any accurate 
means of predetermining baking value. It therefore becomes neces- 
sary to continue the studies of the various factors which go to make 
up this property of flour strength, not by themselves, but relative- 
ly and collectively. 

Biochemical Method. — The wheat berry containing the plant em- 
bryo and associated food stored there for nourishment of the plantlet 
is produced by the plant for the propagation of its own species. The 
commercial flours produced by the roller milling process, and known 
as patent, straight, first and second clears, contain about 70 to 80 per- 
cent of the wheat berry, mostly of the food storage material, with 



increasing amounts of the branny covering's and fragments of em- 
bryo, in the order named. Any differences in the laying down or 
"setting" of this storage material, or in the development of the em- 
bryonic elements, were imposed by the biological changes during 
growth, ripening, and storing of the wheat, and will, therefore, be 
reflected in the cjuality of the flour after milling. Studies on the 
progressive development of the wheat kernel by Teller (1898), Wym- 
per (1909), Brenchley and Hall (1909). Thatcher (1913, 1915), Eck- 
erson (1917) ; and those on the chemical changes in wheat under 
various conditions of storage or handling by Swanson (1916), Olson 
(1917), Bailey and Gujar (1918-1920), and Blish (1920), will justify 
our belief in the dependence of flour quality vipon biological bases. 
The problem of flour strength must, therefore, be approached from 
the bio-chemical point of view: first, to gather all the available in- 
formation concerning the character and properties of each individ- 
ual biological factor contributing to flour strength ; and second, to 
study these factors relatively and collectively in their many and labile 
relationships. The factors to be controlled in such an undertaking 
appear to be almost without limit, and, therefore, the task of a collective 
study of flour strength to be almost hopeless. Yet by judicious se- 
lection of constants and variables, a beginning is made which au- 
tomatically leads to a better knowledge of the relationships of pro- 
tein content, gluten quality as shown by viscosity and other colloidal 
properties, enzyme activity, carbohydrate content, gas retaining abil- 
ity, yeast activity and fermetation, effect of shortening agents, acid- 
ity, buffer action of salts, chemical "improvers" and yeast stimulants, 
temperature, time, etc. 

Purpose — While this investigation was undertaken primarily to 
set aside and study in detail the diastatic enzymes of flours in their 
relation to flour strength, the plan also included the bringing to- 
gether of as many controlled factors as possible to bear on a series 
of selected flours. The data published by earlier workers had been 
largely confined to the study of one or two factors on a limited num- 
ber of flours, largely because of the enormous expenditure of time and 
energy required to make a more collective study. These flours can- 
not later be duplicated. And inasmuch as flours differ so widely 
among themselves it has been difficult for another worker to take 
up the problem and secure comparative results even when the same 
technique is employed. This applies even more strongly when at- 
tempts are made to correlate data by different workers on different 
factors. It was hoped to overcome this difficulty to some extent in 
the present work, first, by collecting fifteen samples of flour milled 
from authentic samples of wheat and representing various typical 



wheat producing areas of North America ; second, by the cooper- 
ative work of three investigators working on the same set of samples 
but from different points of attack; and third, by a complete record 
of all the available analytical data obtained on each flour. The co- 
operation of the American Institute of Baking research laboratories 
with the Division of Agricultural Biochemistry of the University of 
Minnesota made such a plan possible. The work on this series of flours 
has now extended over nearly two years. The present paper is the first 
report. Additional factors wlill be considered by the other col- 
laborators in subsequent publications. 

HISTORICAL 

Payen and Perzos in 1833 proposed the term diastase to designate 
that active agent in malt which transformed starch to dextrins and 
some form of sugar. Jago (1911) further characterized as diastase 
that agent of malt which transforms starch or soluble starch to mal- 
tose. In the voluminous literature on the subject of amylase and dias- 
tase, the terms are sometimes used interchangeably. Later tendency 
is to distinguish between amyloclastic and saccharogenic action de- 
pending upon which activity is to be measured. Unfortunately this 
differentiation has not been as general as might be desired. 

Definition of Diastase. — LeaA'ing a consideration of the character 
of the two enzymes involved for later discussion, it should be stated 
here that the term diastase is restricted throughout this paper to 
mean "That enzyme or group of enzymes which through their suc- 
cessive or cumulative action produce maltose from the starch avail- 
able." 

It is impractical to consider in detail the great mass of literature 
on the subject of diastase in the malting and brewing industries ex- 
cept as it applies directly to the problem of better bread. 

In 1907 T. B. Wood published the results of some studies on the 
the factors influencing the size and shape of the loaf. His paper 
marks the transition from the purely chemical analytic to a biological 
point of view. Although a sharp distinction was not drawn between 
the amount of sugars in the flour and those produced by diastatic ac- 
tion, he clearly recognized "that the sugar so formed, togetherwith that 
originally present, forms the source from which the yeast makes the 
carbon dioxide it produces when the dough is fermented." This re- 
fers to the baking process then prevalent in England in which little 
or no added sugar was used in the dough batch, the fermentation 
depending almost entirely upon the sugars produced by diastatic 
action. To arrive at some measure of the sugars thus available for 
yeast fermentation, 20. grams of flour with 20 cc of water and 0.5 

10 



grams of yeast were incubated at 35 °C, and the volume of carbon 
dioxide given off was measured over a period of hours. The result- 
ing data led to the conclusion that: "Here too the rate of gas evolu- 
tion and the size of the loaf run parallel, and it seems certain, there- 
fore, that it is more particularly the gas given ofif in the later stages 
of dough fermentation that determines the size of the loaf. This 
being so the size of the loaf will depend, not so much on the sugar 
present in the flour as such, as on the diastatic capacity, which will 
cause continued sugar formation, and consequently continued gas 
evolution in the dough. Probably, therefore, measurement of the gas 
evolved in the later stages of the fermentation would give a more 
accurate test for the power of making a large loaf than the measure- 
ment -which I have made of the total volume given off in 24 hours." 
Shutt (1907) in commenting on Wood's paper, expressed a belief, 
founded on analysis of sugars in flours, that diastatic activity rather 
than the sugar content in a flour is the determinative factor in loaf 
volume. His limit for reducing sugar is .62% and for non-reducing 
sugar 1.22%. A few months later there appeared the contributions 
of Baker and Hulton (1908) and Ford and Guthrie (1908). These 
two investigations, made independently of each other, were the first 
attempts at a systematic study of the enzymes of wheat flour in 
their relation to baking value. Ford (1904) had previously considered 
the estimation of diastatic power in malt flours, using soluble starch 
as a substrate. Many necessary precautions were pointed out con- 
cerning the preparation of pure soluble starch, and the effect of 
temperature, time, and acidity on the extraction of diastase, so that 
the results of this oft quoted article have an important bearing on 
the determination of diastatic activity in wheat flour. 

Baker and Hulton's statement of the importance of diastase action 
in fermentation is in agreement with that of Wood as quoted above. 
They said "It is certain that some of the carbon dioxide concerned 
in the rise of bread, especially in the later stages of doughing and 
in the earlier period of baking, is formed from the fermentation of 
the maltose produced by the action of the diastase on the flour 
starch." They demonstrated the presence of diastatic action in a 
dough by extracting the sugars and preparing the osazones of 
glucose, and glucose and maltose, respectively, from two doughs 
made without yeast, one of which had the diastase inactivated by 
.02N NaOH, the other allowed to remain active for four hours at 
40°C. But their measurements of the activity of flour extracts on 
soluble potato starch, although indicating its similarity to barley 
diastase (Baker, J. L., 1909), showed that large differences exist 
between this activity of extracts and the activity of the enzyme-con- 

11 



taining tissues when in the state of a dough, and it was recognized 
that this work on soluble starch, after the method of Lintner, gave 
no measure of liquifying enzymes. They found by measuring the 
amount of carbon dioxide produced during fermentation of a dough, 
as suggested by Wood, that there was a close correlation of carbon 
dioxide production to maltose resulting from diastatic activity, cor- 
recting for original sugars present. They also pointed out, however, 
that "a weak flour may have a diastatic power as high as or even 
higher than a strong flour." 

The conclusions drawn from data on carbon dioxide production 
are of especial interest because with the exception of the few trials 
by Olson (1917), no results have been obtained for the liquifying 
agent in diastase of wheat flour. To quote from his conclusions : 
"The results indicate, we think, most conclusively that the low gas 
production of this flour (a weak flour) arises fron an inadecjuate 
supply of starch liquifying enzyme. We have already shown that 
the gas to diastase ratio is higher on the whole in strong flours, and 
it seemed probable, in view of the last experiment, that we might 
establish a connection between the strength and the relative amount 
of a starch liquifying enzyme in a flour." 

The same phase of this problem was approached from a different 
angle by Ford and Guthrie (1908). Most of their efforts were directed 
toward obtaining complete extraction of the diastase from flour by 
applying the methods previously used for barley diastase. The eft'ect 
of both added active proteoclastic enzymes, such as "papaien," and 
of various concentrations of added salts, such as sodium chloride, 
potassium chloride, and phosphates, were tried, resulting in 
the increase of diastatic activity in the extract. They state : "The 
amylase of wheat, like highly purified preparations of amylolytic 
enzymes from other sources does not exhibit its full hydrolytic 
activity .except in presence of a certain salt concentration." This 
same principle of salt eft'ect in the protection of diastatic action 
was made use of by Sherman and Walker (1917) and Sherman, 
Thomas and Baldwin (1919) in their studies on the amylases of ani- 
mal, plant and fungus origin. 

The data of Ford and Guthrie for the diastatic activity of their po- 
tassium chloride extracts from twelve different flours showed but 
slight differences in value as maltose per gram of flour. They carried 
autolytic measurements further than Baker and Hulton had done, 
but with the object of determining what effect the proteoclastic ac- 
tion would have on the diastase. After allowing the 1 : 25 flour water 
suspension to digest autolytically for different periods of time at 30°C. 
the diastase was extracted and its action on 2% soluble starch was 

12 



measured. The resulting- data which are presented here as Table 
1 shows the peculiar fact that such a procedure gives practically 
a constant diastatic activity regardless of time. The results of That- 
cher and Koch which are discussed later, showed similar results 
with the same sort of treatment. 

TABLE I. 
"Autodigestion of Wheaten Flour" 
(The digestion of soluble starch by diastase extracted from flour- 
water suspensions which had been allowed to digest autolytically for 
different periods of time): (Ford & Guthrie, 1908). 

Grams Maltose per 1 g. Flour 
Flour No. 1 Flour No. 2 

After 1 hour 2.87 11.83 

After 3 hours 2.87 11.90 

After 4 hours 2.80 11.69 

After 5 hours 2.66 11.34 

After 26 hours 2.52 12.74 

One noteworthy suggestion in this contribution is that perhaps the 
degradation of the gluten in weaker flours by the proteases in some 
diastatic malt preparations is the reason for their failure to improve 
the quality of the loaf. Confirmation of this hypothesis still awaits 
further research on the proteases of wheat and malt flours. Pre- 
liminary investigations along this line in this laboratory point to the 
undoubted importance of proteases as factors in panary fermentation. 

Baker and Hulton also had considered the proteoclastic enzymes 
of the flour, but expressed the opinion that these enzymes do not ex- 
ert sufficient effect on the gluten during the baking period to be of 
serious consequence in the baking value of a flour. On the other 
hand, however, they point out that the proteoclastic enzyme of the 
yeast probably play a more important part in the modification of 
the gluten during fermentation. This fact was demonstrated by the 
presence of 2.7% of soluble nitrogen (as protein) in a fermented 
dough as compared to 1.9% in the same dough made without 
yeast. Liebig in 1909 reported the results of comparative sugar de- 
terminations made on wheat meal and on doughs made of the same 
meal. He found that maltose was steadily produced by the action of 
diastatic enzymes in a dough after standing fourteen hours at 30 to 
40 degrees, and the reducing sugar content calculated as dextrose 
amounted to 4.6 percent. Doughs fermented two hours with yeast 
showed an excess of unfermented reducing sugar which Liebig con- 
cluded was due to the action of diastase. 

There was a great deal of general interest shown by the chemists 
at that time in the diastatic and proteoclastic enzymes of wheat 
flour. Neumann and Salecker (1908), and Kohman (1909), demon- 
strated the improvement of bread by the addition of active dias- 

13 



tatic malt preparations. Humphries (1910) stated very clearly the 
position of diastase as a factor in flour strength. Armstrong (1910) 
likewise discussed its practical importance in the bakery but he 
doubted the possibility of correlating diastatic power with flour 
strength. Armstrong objected to Wood's (1907) measurements of 
gas production and diastatic power because "they were made under 
conditions very different from those which prevail in actual bakehouse 
practice." 

~ Most of the earlier data in the literature, while significant, is of 
less value than its accompanying discussion. This is true chiefly 
because of the difficulty in controlling the conditions necessary for a 
study of the wheat flour enzymes. 

Sherman, Kendall and Clark (1910), reviewed the various methods 
for use in the determination of diastatic power as a preliminary to a 
careful series of studies on the amylases of different origin. The 
method and the scale adopted by them depended upon the weight of 
maltose produced by the action of the diastatic preparation on soluble 
starch at 40°C for 30 minutes. Sherman's prediction (1910) that 
the determination of diastatic power would "soon become an impor- 
tant factor in the valuation of commercial American Malts," has 
become a fact. The registration of diastatic malt preparations for 
sale to the baking trade has shown the necessity for practical, uni- 
form, standard methods of measurement. (American Institute of 
Baking, 1921.) The measurement of diastatic power as carried out 
at the present time among the control laboratories of this country 
show the most surprising variations in method and result. The Lint- 
ner method (1886). or one of its modifications, is still the basis for 
most of them. 

The most important data in the literature to date concerning the 
autolytic diastatic activity in wheat flours is that of Swanson and 
Calvin (1913). They stated: "If flour itself possesses such large di- 
astase activity that by digesting it with water at a suitable tempera- 
ture, more than one fifth of its weight is transformed into soluble 
carbohydrates such as glucose and maltose, the conditions for such 
transformations deserve careful study." These authors allowed the 
diastase of the wheat flour to act autolytically on the wheat starch 
in water suspensions of various concentrations, measuring the amount 
of maltose produced. This procedure would appear to give an index 
of the activity to be expected in a dough. But no use has been 
made of this valuable data in the improvement of baking science. 
It is now impossible to duplicate the flours they used ; nevertheless, 
the data can be ognsidered as quite comparable to that obtained on 
other flours of similar quality, and so may be used on that basis. 

14 



k 



The problem of diastase in the cereal grains was again attacked by 
Thatcher and Koch in 1914. Instead of applying auto-digestion as 
did Swanson and Calvin, they undertook to obtain extracts of constant 
diastatic power by digestion of the ground cereal with water at 
0°C for two hours. This extract was then filtered in the cold, 
allowed to act on soluble starch for 30 minutes at 40°C. and the 
resulting maltose determined. The tables of results show some 
variation with time of extraction, which was due to the loss of dias- 
tatic power with a corresponding increase of sugars even at the low 
temperatures employed. Comparative results for one to five hour 
extractions could only be obtained because the time curve for dias- 
tatic action at 0°C rises so slowly that differences between one and 
three hours were within the experimental error of the methods em- 
ployed (Fig. 2). 

The probable error of a determination based on 30 minutes action 
at 40° C is large, because this point occurs on the steeper part of the 
curve, where differences of 1°C or 1. minute in time is considerable. 
The fact that no significant differences were observed between 
medium and fine grinding of the materials apparently eliminates at 
least one possible variant in the study of cereal diastase. Their 
results confirm the precautions necessary in the determination of malt 
diastase as pointed out by Kjeldahl, Ford, Brown and Glendening, and 
others, namely, there must be present so large an excess of starch 
that less than 40% is hydrolyzed to sugars. 

Swanson, with Fitz and Dunton, published in 191(3 the results of 
milling and baking tests on wheats which had been subjected to 
different methods of handling and storing. The eft"ect of increased 
diastatic enzymes by the addition of small amounts of germinated 
wheat flour was to increase the "spring" in the oven and increase the 
volume of the loaf. Too large amounts, or flour from wheat which 
had germinated over too long a period, resulted in a weakened 
gluten, poor texture, and an inferior loaf of bread. More conclusive 
data was published the next year by Olson (1917), who made com- 
parative baking tests on strongly diastatic flours milled from germin- 
ated wheat. No measurements were made on the relative saccharo- 
genic powers of these flours, but their amyloclastic powers were com- 
pared by incubating the 1 : 20 flour-water mixture at 70° C. until the 
iodine test failed to show the characteristic blue color. The increase 
of amylase activity in the flour from germinated grain with a one 
centimeter epicotyl as compared to that from grain which had only 
started to germinate, was remarkable. Loaves baked from the germ- 
inated wheat flour showed increases in volume, but those containing 
too much amA'lase suftered in texture and character of crumb because 

15 



of too great liquifying action. This dextrinizing action supports 
Humphries' (1910) contention that added diastase in small quantity 
had the property of very perceptibly improving the flavor of a loaf, 
probably due to the correlation of increased dextrin content and the 
consequently improved conditions for the retention of moisture within 
the loaf. Olson showed further that the addition of varying small 
quantities of these diastatic flours to a normal flour did increase the 
water-holding capacity in direct proportion to the amount of germin- 
ated wheat flour added. In one case the volume of the loaves were 
increased as much as 49% without apparent impairment of the quality 
of bread, and in all cases there was an increase in volume, but the 
important fact in this connection depends upon the inherent "strength" 
of the normal flour used. The gluten of some weaker flours is of 
such poor quality that it cannot retain the extra gas produced through 
the agency of an added excess of diastase, and consequently the tex- 
ture of the loaf suft'ers. It appears from those results that added 
diastase within certain limits, improves the baking quality of a flour, 
but the inherent strength of the flour governs the quantity of diastase 
which may be used. These conclusions agree with the work of 
Humphries, Kohman, Neumann and Salecker. 

The data on the variations of the dififerent forms of nitrogen com- 
pounds in the germinated wiieat flours also lead one to suspect that 
perhaps the proteoclastic enzymes, in their degradation of the gluten, 
have had a more profound influence in the impairment of the grain 
and texture of the poorer loaves than the action of the diastase. Con- 
firmation of this probability must await further research on the ac- 
tivity of proteoclastic enzymes and their effect in baking. The recent 
work of F. J. Martin (1920) is in effect a continuation of the investiga- 
tions of Wood (1907). The volumes of gas given oft" by fermentation 
of 20. grams of flour with 55% water, 1.2% salt, and 1.0% yeast, over 
a period of 24 hours at 29° C were measured at dift'erent points of time. 
The volumes of the dough were in close relation to the gas production 
during the first periods of fermentation. But after a normal fermen- 
tation period of about three hours the relation was complicated by 
the reduction . of the gas retaining power of the dough, due to a 
weakening of the gluten. There are several points in Martin's paper 
which are of significance in a study of enzymatic activities in flour. 
The variation in the volumes of gas produced by the different flours 
at any stage of fermentation was not great, and could not be cor- 
related closely with final loaf volume. But in the case of a weak 
flour, one which showed a low gas-producing power, the deficiency 
of gas production in the later stages of fermentation could be rectified 
by the addition of diastatic enzymes, with a consequent increase in 

16 



volume of the loaf. These results are in agreement with those of 
Kohman and Olson. The anal3''tical data on Martin's flours ofifer 
some interesting comparisons. They would appear to show the pos- 
sibility of obtaining a constant for the ratios between water soluble 
proteins, gas retaining powers, and bakers' marks. The difference 
between his gliadin figures and the so-called "amended gliadin" point 
to a possible basis for the rough measurement of proteoclastic activity, 
since the water soluble protein increased with length of extraction 
period, at the expense of the alcohol soluble protein. In the second 
paper Martin (1920), demonstrates an increase of gas producing 
capacity, e. g., diastatic power, with the increasing percentage of wet 
and dry gluten as the source of the flour progressed from the center 
of the endosperm outward to the cortex. This data would appear 
to corroborate the opinion of Teichek (1904), that diastase, while 
largely concentrated in the germ, is also distributed throughout the 
endosperm and extends throughout the wheat berry. The studies of 
Whymper (1909), Mann (1915), and others, on the other hand point 
to the embryo as the only source of diastase in the grain. A further 
examination of these results, with analyses of both diastatic action 
and nitrogen partition might lend support to the postulate that the 
diastase of the wheat berry is associated with, or held by the protein 
body and functions normally only in their presence. Also that the 
peptizing action of the proteoclastic enzymes on the proteins has 
some bearing on the action of the diastase. 

The difficulty of using the expansion volume of a dough as a 
measure of diastatic capacity is due to the weakening of the gluten 
after the second or third hour. "Curves plotted to show the relation 
between the amounts of gas generated and the volumes of the doughs 
w^ere fairly regular for the first part of the experiment, but erratic 
for the latter part." (Martin (1920). The recent work of Bailey 
and Weigley (1922) afford a more complete summary of this relation- 
ship. 

PRELIMINARY DISCUSSION 
There are a number of factors which limit the production of maltose 
in a dough by the agency of diastase. 

1. Liquifying and Saccharogenic Action. — Baker and Hulton 
(1908) believed that the diastase of grains contained both a liquifying 
and a saccharogenic part so that the amyloclastic power would become 
the limiting factor in maltose production. This idea is frequently 
expressed in the literature on diastase in bread making, but to date 
no satisfactory methods have been developed to accurately measure 
it. Nor has that phase of the problem been undertaken in this inves- 
tigation for reasons which will ajipcar further on in this discussion. 

17 



The presence of these two distinct activities in malt and wheat dias- 
tase have nevertheless been well established. 

Without attempting to analyze the numerous but inconclusive data 
on the identity of the two distinct activities in diastase, it is suf^cient 
to recognize their interpendence in the production of a fermentable 
sugar from natural starch, and summarize the later work on the sub- 
ject. Sherman and Schlesinger (1913) made comparative measure- 
ments of the amyloclastic and saccharogenic power of the malt and 
pancreatic preparations. Their paper ofifers a comprehensive list of 
references on the distinction between the two kinds of activity, but 
no conclusions are drawfn as to the identity of the separate enzymes.- 
While their results on pancreatic amylase indicate a fairly constant 
relationship between the amyloclastic and saccharongenic activity of 
their pancreatic preparations, the same method of measurement failed 
for the malt preparations, the amount of starch liquified apparently 
being less than the maltose produced therefrom. The explanation 
suggested to account for the observed discrepancy was : That these 
two activities are characterized by dilTerent conditions of optima for 
their respective rates, i. e., of temperature, acidity, and salt concen- 
tration. Sherman and Thomas (1915) supplied further evidence in 
support of that explanation by their measurements of the optima 
for amyloclastic and saccharongenic actions. Whether this explana- 
tion be the correct one, and the two distinct activities be due to 
separate, conjunctive enzymes; or whether the special properties of 
organic colloidal catalysts can perform the two degrees of hydrolytic 
power under the influence of changing conditions in media ; it should 
be possible to more accurately characterize the amyloclastic and sac- 
charogenic parts. To mention only a few of the possibilities, it would 
appear that the researches of Sorenson (1917). Robertson (1920), 
Meutscheller (1920), and Warden (1921), in the application of phys- 
ical and electro chemistry to biological problems, furnish many meth- 
ods which might be applicable to the separation and identification of 
these two enzymes if they exist as such, or to the establishment of 
the identity of the catalyst responsible for the different degrees of 
hydrolysis. The remarkable results of Warden add another link in 
the chain of accumulating evidence for the hypothesis that enzymic 
activity is but another manifestation of the catalytic process function- 
ing by virtue of those surface forces which are active in colloidal 
particles. Such studies are obviously beyond the compass of this 
set of experiments and pertain more to the theory of enzyme action. 

In the methods of Sherman and Schlesinger noted above, soluble 
(gelatinized) starch was again used for the measurement of both 
amyloclastic and saccharogenic power, and therefore cannot be 

IS 



applied directly to the question of amyloclastic activity as the limiting 
factor in diastatic action in the dough. 

2. Resistance of Different Starches to Diastatic Action, It is only 
the final production of maltose, resulting from the diastatic activity 
of a given flour, which is of consequence to the baker, and the various 
limiting factors, including the amyloclastic activity and the resistance 
of that particular starch, are summarized in that final result. Yet 
when a method is applied also to the measurement of diastatic powers 
of malt preparations, which are intended to act on the natural starches, 
the question of the resistance of the natural wheat starches becomes 
of first importance, both from the standpoint of the final product, and 
for the selection of a standard substrate. 

There appears to be a difference in the condition of the starch in 
the various kinds of flour. The difiference was noticed by us in 
working with suspensions of the flour samples under investigation. 
Whymper (1909) by microphoto examination of starch, showed dif- 
ferences in the resistance of difl:erent starch granules in flour to dias- 
tase. Simpson (1910) "has shown that under certain conditions a 
small proportion of flour converted into sugar a quantity of ungelatin- 
ized starch equal to 8% of the weight of the flour, but that under 
identical conditions the same quantity of the same flour converted 
a quantity equal to 400% of its own weight into sugar when gelatin- 
ized starch was used." Stone (1897). was of the opinion that there 
were difterences in the action of enzymes on starches of difterent 
origin, but Ford (1904), confirming O'Sullivan's previous observa- 
tion (1904), concluded that there is practically no diiTerence in the 
action of diastase on starches even of different origin under com- 
parable conditions of acidity and temperature. Their data, however, 
was obtained on modified, or "soluble" starch, and so may not be 
applied to the question of the biological differences in natural wheat 
starch. Sherman, Walker, and Caldwell (1919) also reached the con- 
clusion that there was little or no dift'erence between the resistance 
of different starches to the same diastase preparation, but their 
starches had likewise been boiled. The important work of Reichert 
(1916), and of Dox and Roark (1917). would lead one to expect a 
variation in the resistance of starches of different origin to the action 
of enzymes. Stakman (1918) and Leach (1919) have pointed out 
the significance of resistance to parasitism which is shown by different 
varieties of wheat. Considering the penetration of parasites into the 
host as the result of enzymatic action, the variable resistance of dif- 
ferent biological conditions to enzymes may well be expected to 
show in the starch granules as well as in other plant tissues. The 
recent articles by Samec and his co-workers (1921) present some 

19 



extremely interesting possibilities in the chemical, physical, and elec- 
trometric differentiation between starches of different biological 
origin. But the susceptibility of biologically different samples of 
wheat starch to diastatic activity is but one of the controlling factors, 
and like the other factors, is reflected in the resulting degree of dia- 
static power. 

3. Difference Between Autolytic and Extracted Enzyme Activity. 
In the wheat berry the diastase is laid down by the plant for the 
purpose of transforming the starch granules of the endosperm into 
soluble sugar available for assimilation and growth of the young 
seedling. Whether or not in the economy of the seed there is a 
separate preparatory or liquifying enzyme, a cytase which prepares 
the starch granule such as suggested by Armstrong (1910), and makes 
it susceptible to the hydrolytic action of the diastase (Wallerstein 
1917, and others), the result is the same. At any rate during panary 
fermentation, the diastase of the flour, and likewise any diastase 
added to the dough in the form of malt flour, malt extract, or other 
diastatic preparation, must produce maltose from the wheat starch 
as it exists in the flour, unless other forms of starch be added to the 
dough. 

The results of Ford and Guthrie (1908), have indicated that the 
greater enzymic activity was obtained by auto-digestion of malt, and 
by addition of salts and proteoclastic enzymes, those of Baker and 
Hulton (1908) that flour extracts do not furnish a true measure of the 
diastase present in the flour; those of Sherman and Baker (1916), that 
purified malt extracts show dift'erent activities on different forms of 
starch substrate (prepared), and those of Swanson and Calvin (1913) 
have demonstrated the value of the autolytic method for flours. Yet 
the methods in general use at present for the measurement of diastatic 
power all depend upon the action on "soluble" starch. In other 
words, an artificial and by no means standard substrate, whose col- 
loidal characteristics have been profoundly altered, is the starting 
point for an arbitrary procedure, the result of which often has no 
bearing on the activity intended to.be measured. It was mentioned 
above that the same diastase acting on soluble or gelatinized starch 
may produce several hundred times as much maltose as when allowed 
to act on raw or unbroken starch kernels. It will be demonstrated 
later, (pp. 72, table XXII) that this is a very real source of error in 
the determination of diastatic power of different diastase preparations. 
Thatcher and Koch also found that extracts prepared by their method 
gave lower diastatic values than were obtained in equal aliquots of 
unfiltered extracts from the same flour. A little later "attempts to 
apply the method in a comparative study of the diastatic activity of 

20 



wheat flour of different grades and various processes of manufacture 
gave the surprising result that extracts of approximately uniform dias- 
tatic qualities were obtained from flours of widely varying character 
and baking qualities."* Still more recently R. W. Thatcher and Cor- 
nelia Kennedy (1917) obtained some valuable data bearing on the loss 
of diastatic activity in flour extracts by various methods of treatment.** 
One series of flour samples were subjected to auto-digestion with 
water (1 :4) for 1 hour at 0°, and the filtered extract allowed to act 
on soluble starch at different temperatures, as in the proposed method 
of Thatcher and Koch ( 1914). The following table shows the surpris- 
ing difference in result: 

TABLE II. 

The difference in diastatic activity between auto-digestion and the 

action of extracted diastase on soluble starch. 

Cuprous Oxide Produced by Diastase 
from .5 Grams of Flour 





Auto-digestion of 


Extracted 1 hr. @ 0°C 




Flour-water 


(1:4) 


Fihered Extract on 


Temperature of 


1. hour 




Soluble Starch 1 hr. 


Action on Starch 


Grams 




Grams 


40° 


.02390 




.08485 


50" 


.05497 




.08125 


62° 


.15296 




.08050 



It was likewise found that the unfiltered extract acting on soluble 
starch at 40, 50 and 62° C showed 1.673 g. Cu. per .5 g. flour as com- 
pared to only .4302 g. Cu. per .5 g. flour for the filtered extract; a loss 
of about 74% of its activity through filtration. An examination of 
the residue on the filter would indicate that the tenacious glutinous 
mass had adsorbed some of the enzyme, or else had held back an 
activator. This residue was apparently not tested directly for its 
activity. But samples of gluten were washed out in the usual manner 
and wiere kneaded with distilled water until no more starch could be 
removed. After dispersion in N/200 lactic acid their diastic activity 
on soluble starch was compared with an extract, both filtered and 
unfiltered. The results are given in Table III. 

TABLE IIL 

The relative diastatic activity of water extracts and of gluten 
from the same flour. 

1. 0.895 g. Cu. per .5 g. flour (Filtered extract on soluble starch) 

2. 3.200 g. Cu. per .5' g. flour (Unfiltered extract on soluble stardh) 

3. 0.818 g. Cu-. per .5 g. flour (Gluten dispersed with N/200 lactic) 



*(Geo. P. Koch's unpublished results in a thesis presented to the Faculty 
ot the Graduate school of the University of Minnesota, in partial fulfilment 
of the requirements for the degree of Master of Science). 

**(Grateful acknowledgment is hereby made to these authors for their 
permission to publish the results of their experimental data in this connection). 

21 



It would thus appear that the gluten had retained nearly half 
of the diastatic activity. Unfortunately there is no data available 
whereby a correction can be applied for a possible activating influence 
of the N/200 lactic acid. This absorption of enzyme by gluten was 
further examined by extracting 4. g. flour with 100 cc. H2O, (in which 
were dissolved .006 g. takadiastase) for 1 hour @ 0°. Part of the 
extract was filtered and compared to the unfiltered portion in its 
action on soluble starch. The unfiltered extract produced 6.725 g. 
Cu. as against only 3.215 g. Cu. for the filtered extract. Baker and 
Hulton have found and recorded that some diastatic activity was 
shown by the glutens thus washed out of wheat flour. 

In the course of these experiments of Thatcher and Kennedy it was 
desired to ascertain whether only maltose was produced by diastasis. 
The increase in Fehling reduction by inversion of the solution after 
diastatic action had been stopped was measured. 

TABLE IV. 

The relative reduction of Fehling solution before and after inversion 

of the sugars produced by diastatic action. 

Before Inversion After Inversion 

grams cu. grams cu. 

I. .02208 .04416 

II. .03940 .06760 

III. .02784 .05376 

IV. .03264 .06192 

In spite of the evidence of Sherman and Plunnett (1916), for the 
production of small amounts of glucose in addition to maltose by malt 
amylase, the fact that maltose is the only sugar produced by diastase 
in sufficient quantity to be of any significance in panary fermentation 
has been recently confirmed in this laboratory. (Collatz, 1922). 

One peculiarity of the results obtained by Thatcher and Kennedy, 
however, still remains to be explained. The diastase activity in flour 
suspensions has been shown by Swanson and Calvin, and confirmed 
here, to be extremely sensitive to temperature, the curve appearing 
autocatalytic in character up to nearly 60° C. Yet the activity of the 
filtered extracts of Thatcher and Kennedy showed the same value 
whether acting on soluble starch at 40°, at 50, or at 62°C, while the 
unfiltered extract showed a temperature effect. There are obviously 
several possible explanations: (1) The filtration may remove some 
substance which functions as an activator (Thatcher, 1921); (2) 
The proteins of the flour may absorb some essential factor in the 
diastatic action, and; (3) the contributing, perhaps controlling, effect 
of the phosphates and other buffer salts present in the flour (Bailey 
and Collatz 1921, and Bailey and Peterson, 1921), do not function 
under those conditions of extraction and measurement. Neither of 

22 



tticse explanations seem to be sufficient. The enzymes are un- 
doubtedly colloidal in character. Their hydrolytic acti\it}-, involved 
as it is in the complex properties of colloidal material such as surface 
action, adsorption, dispersion, etc., is, therefore, extremely sensitive 
to any changes in the electro-chemical and physical nature of the 
media in which they are acting. 

It has been found that the starch as u-ashed out of flour carrie= 
considerable diastase, the gluten retaining by far the greater portion 
Removal of the protein matter from the starch granules by dispersion 
and repeated washing effects the removal of the diastase, indicating 
that the diastase is neither adsorbed to nor associated with the starch. 
K. Mohs (1920, 1921), has presented interesting expositions on the 
colloidal theory of diastatic activity and though probably carried 
further than experimental facts warrant, they are extremely sugges- 
tive for experimental verification. It appears certain, in short, that 
extraction of diastase from biological material, with subsequent filtra- 
tion and hydrolysis of modified starch by the filtrate, will not alTord 
an accurate conception of the true activity as it exists au nature!. 
Therefore, any practical method for the measurement of the value of 
diastatic enzymes in baking must be based upon condlt'ons as they 
exist in the dough. The increasing general use of diastatic prepara- 
tions in baking practice for the improvement of bread makes it im- 
perative that a new procedure be developed. It must give a measure 
of the ability of that diastatic preparation to produce sugars which 
can be utilized by the yeast for growth and carbon dioxide production. 

EXPERIMENTAL 

A survey of the literature on diastatic activity has shown the 
unquestionable importance of these enzymes in panary fermentation. 
The fact that the bakers in America are estimated to use something 
over thirty million pounds of malt per year, a large part of which is 
diastatic in character, with a probable valuation of over two and a 
half million dollars, is sufficient evidence of the economic interest in 
the problem. The relative importance of this activity as a factor in 
the composite strength of wheat flours has not been settled. The 
interest of the baker in this question has steadily grown until a satis- 
factory answer should contribute materially to baking science. 
Consequently the general use of diastatic preparations for the im- 
provement in volume and flavor of bread, and the numerous but in- 
conclusive contributions found in the literature on the effects of dias- 
tase in panary fermentation, have all served to demonstrate the great 
desirability of further research on the diastatic enzymes of wheat 
flour in their relation to flour strength. 

23 



The Materials. To obtain data which would furnish the basis for 
conclusive evidence as to the role taken by diastase in dough 
fermentation, it was considered necessary t o obtain s a ni pies 
of wheat flours of widely varying- characteristics. Samples of flour, 
of different grades and baking strengths were accordingly obtained 
from eight of the typical bread-flour wheat producing areas of North 
America. These districts include the Northern Great 'Plains area, the 
Washington Walla Walla, the Saskatchewan and Alberta Canadian, 
the Kansas, the Utah irrigated, the Montana Dry Farming and the 
Ohio wheat areas. The Sitka, Alaska mills were not yet completed 
and the Alaskan wheat sample obtained was too small to furnish suf- 
ficient standard flour for comparison with the other samples. The 
most of these samples of flour, in fifty pound lots, were obtained di- 
rect from the commercial mill in which they were ground. In the 
case of samples Nos. 1012, 1013, and 1014 the wheat was obtained 
from Fergus county, Montana, and was selected to represent the aver- 
age wheat as raised in the district. It was shipped direct to Fargo, 
N. D. and there reduced to flour at the experimental mill operated b> 
the Agricultural Experiment Station of North Dakota. In all cases 
samples of the wheat from which the flour was milled were kept for 
further check and examination. Thus the flours studied in reference 
to their diastatic activities are known to be representative of the wheat 
growing districts from whence they came. It should be remarked, 
however, that three of the samples, number 1001, 1002 and 1009 are 
milled from blended wheats. Numbers 1001 and 1002 are patent and 
1st clear respectively, milled from a regular "mill mix" and are typi- 
cal of the flours generally milled in that locality. Number 1009 should 
receive special mention because of its relation to the other flours. It 
is a well known commercial brand of high grade patent flour, milled 
from a two-wheat blend in the Northwest, and was selected because 
of its superior quality. The composite of its "strength" characteris- 
tics, as well as its combination of qualities as shown in the baked loaf, 
gave it the highest baking value of any of the flours available. Con- 
sequently this sample was used as a standard against which to com- 
pare all others. 

The objection might be raised that these samples, numbering four- 
teen in all, do not furnish sufficient bases of seasonal and climatic va- 
riation for a complete characterization of enzymatic activity in rela- 
tion to strength. Yet it is believed that sufficiently characteristic 
data has been obtained to justify certain conclusions and to furnish 
the foundation for further investigation into the behavior of these 
biological catalysts of wheat flour. 

A history and description of the flour samples follows : 

24 



TABLE V. 



History and Description of Flour Samples. 



Sample 
No. 

1001 

1002 

1003 

1004 

1005 

1006 

1007 

1008 

1009 
1010 

1011 

1012 

1013 

1014 



Locality 
(Where Grown) 



Type 
Turkey Red 
Turkey Red 
Little Club 



-Wheat- 



Flour Grade 



Reno County 

Central Kansas 

Reno County 

Central Kansas 

Washington 

Walla Walla 

Red River Valley Marquis and 

North Dakota Bluestem 

Red River Valley Marquis and 

North Dakota Bluestem 

Canada Marquis 

Southern Alberta 

Canada Marquis 

Southern Alberta 

Canada, Saskat. Marquis 

Valley, Saskatoon 

Red River Valley Mostly Marquis 

Utah Like Sonora 

Irrigated Valley California 



Class 
Hard Red Winter Patent 
Hard Red Winter 1st Clear 
White Club Straight 

Hard Red Spring 2d Clear 
Hard Red Spring Patent 
Patent 
1st Clear 
Patent 



Selected Hard 
Red Spring 
Selected Hard 
Red Spring 
Hard Red Sprin< 



Hard Red Spring Patent 
Straight 



Ohio 

Williams County 

Montana, Judith 
Basin, Dry Farm- 
ing 
Montana. Judith 
Basin, Dry Farm- 
ing 
Montana, Judith 
Basin, Dry Farm- 
ing 



Soft Red Winter Long Patent 



Wheats 

Resembles 

Fultzo-Mediter 

ranean 
Common Turkey No. 2 Hard Red Patent 
Red Winter 

Common Turkey No. 2 Hard Red 1st Clear 
Red Winter 

Common Turkey No. 2 Hard Red 2d Clear 
Red Winter 



Further notations on the special characteristics of the individual 
samples will be recorded in connection with the experimental data. 

In order to obtain a satisfactory conception of the relative baking 
strength of these flours it was first necessary to make preliminary bak- 
ing tests. These were carried out by the baking expert of the Amer- 
ican Institute of Baking, and under the author's constant surveillance 
and supervision. This baker had had wide experience with all types 
of flours in various parts of the country, and so was especially well 
fitted to bake and judge these loaves. The same individual baked all 
of the flours, in groups of four, repeating the standard and one of the 
other flours for comparison with each succeeding day's bake. The 
method used was that developed for a standard baking practice by 
this Institute for its service and research departments. The purpose 
of this standard baking test was to produce a loaf under carefully con- 
trolled and duplicable conditions which should approach as nearly as 



25 



possible to those obtaining in the average American bake shop. It 
is recognized, of course, that conditions of fermentation in a small 
dough batch must of necessity differ somewhat from those in a thous- 
and pound dough. Nevertheless, it has been found that this differ- 
ence, due to what the baker calls "mass action," can be largely com- 
pensated for by a proper increase in the amount of yeast and a propor- 
tionate allowance in temperature. Experience has shown that the 
behavior of a particular flour, as represented by the fermentation, and 
by the "score" of the finished loaf, can generally be taken by the ex- 
perienced baker as a measure of the baking cjuality of that flour when 
subjected to the conditions of quantity production. 

Baking Test. A description of the apparatus used in the baking 
tests will first be given so as to facilitate the discussion of the pro- 
cedure. The service laboratory was equipped with a ten loaf, electric- 
ally heated Despatch bake oven, equipped with mercury and record- 
ing thermometers, and piped for low pressure steam. The fermenta- 
tion box, 72x24x13 inches, and proofing cabinet, 58x22x13 inches, in- 
side measurements, were electrically heated, with thermostatic con- 
trol, and the proofing cabinet was also piped with low pressure steam 
for controlling the humidity. The one pound pans in which all the 
loaves in this series were baked have the following dimensions : Top, 
eight and five-eighths by four and five-sixteenths inches; bottom, 
eight and one-eighth by three and three-fourths; heignt, two and ouv 
half inches. A small Hobart three speed mixer fitted with a two 
pound bowl, was used for the mixing of all doughs. 

Formula. The standard formula, in terms of a one pound loaf 
dough, is as follows: 

Grams Per Cent 

Flour 325.0 100.0 

Water 179.0* 55.0 

Sugar 10.0 3.0 

Yeast 8.0 2.6 

Salt 5.0 1.5 

Lard 6.5 2.0 

History and Description of Flour Samples. 

*The amount of water added depends upon the absorption of the flour 
employed. The "absorption" was determined in the conventional manner, by 
doughing up 100 grams of the flour and recording the number of cubic centi- 
meters of water required to produce a dough of the proper consistency. It is 
a well known fact that the "absorption" as determined in this manner must 
often be changed, depending upon whether the dough stififens, or slackens 
during the course of fermentation. The absorption values as recorded in the 
subsequent data are those found by actual fermentation to be the most desirable 
for the proper fermentation of each particular flour. This is a departure from 
the custom which has usually been followed in comparative bake tests as 
recorded in the scientific literature, but in accordance with commercial practice 
the baker was instructed to modify the absorptions and fermentation times 
in such a way as to produce the best possible loaf, i. e. to show the greatest 
strength of the flour without the use of any additional ingredients. 

26 



The Baking Procedure. — Flour samples of 650 grams each (required 
lor a two-loaf dough) were weighed out into ten inch mixing bowls 
and set in the fermentation cabinet over night at 27° centigrade. 200 
grams of sugar and 100 grams of salt were weighed out together, dis- 
solved in water, and made up to a volume of two liters. The lard was 
weighed out in 13 gram portions for each dough. The yeast was a 
part of the supply delivered fresh each morning for use in the baking 
school, and showed unusual uniformity in fermenting abiltiy. The 
}east was cut from the center of a one pound cake, and 160 grams of 
this were weighed out thirty minutes before mixing the doughs. This 
yeast was suspended in water in a one liter flask at a temperature of 
27 degrees. l\\ the use of these solutions. 200 cc of sugar-salt solu- 
tion furnished the required 2.5% of sugar and 1.5% of salt for each 
two loaf dough, while 100 cc of the yeast suspension contained 16 
grams of comi)ressed yeast. 

It has been previously found that the 20. grams sugar, 10. grams 
salt, and l6. grams of yeast for each dough displaced just 21.6 cc of 
water. Therefore it was necessary to take this extra volume into ac- 
count and add it to that volume of liquid as calculated from the ab- 
sorption. For example, flour 1008 with an absorption of 60 should 
require 650X60=390. cc of water, so in addition to 200 cc of sugar- 
salt solution and 100 cc yeast suspension, there would be required 
90-1-21.6=111.6 cc more water. The sugar-salt solution and the yeast 
suspension were both brought to approximately 27°, and the extra 
volume of water could be warmed or cooled as a convenient control 
for the temperature of the dough, which was always brought out of the 
mixer at an even 27° C. A half degree rise in temperature was allowed 
for each minute of mixing. To mix, the flour was transferred to the 
bowl of the machine mixer, 200 cc of sugar solution, and 100 cc of the 
well shaken yeast suspension were then added from rapid flowing 
{)ipetts, and the mixer started on the lowest speed. As soon as the 
flour had all been taken up in the dough the lard was added and mix- 
ing continued at second speed to the end of the second minute. The 
dough was then cut down from the revolving arm and mixed with the 
mixing arm turning at high speed for another minute. In this same 
manner each dough received the same thorough mixing of approxi- 
mately the same number of revolutions, and of three minutes dura- 
tion. Each dough was then accurately weighed, set in lightly greased 
bowls fitted with large clock glasses, and placed in the fermentation 
cabinet at 27°C (80.6°F). The average total fermentation time was 
five and one-half hours from mixing to baking, from the time 
of the first "punch." The dough was considered to be ready for the 
first working, variously termed "turning," "cutting over," "knead- 

27 



ing," "knocking down," or "punching," by the appearance of the 
dough surface when indented by the finger. If the outer edges of the 
indentation, instead of filling in again, should show a tendency to sag 
down, after a moment, the dough was considered ready for the first 
punch. This consisted of removing the dough from the bowl, knead- 
ing it lightly five or six times to expel most of the gas, and setting 
again. Considering this as 60% of the total fermentation time, the 
remaining period was divided into approximately 28% and 12% of the 
total time, the second punch following the first in about 50 to 60 min- 
utes, and the dough going to the bench for rounding after a third 
punch about 25 to 30 minutes later. 

The method of handling the dough from the mixing to the oven can 
best be illustrated by an example. Time mixed 9:20 a. m. — ready for 
first punch at 11:40 a. m. ; from mix to first punch 140 minutes. 
Taking that as 60% of total time, the total fermentation period should 
be 233 minutes. Taking 28% of 233, or 65 mintues, the second punch 
would come at 12:45 p. m., and the third at 1:13 p. m. The third 
punch is in reality a rounding up of the dough, corresponding to the 
machine rounder in the modern bakery. After being rounded up the 
doughs were allowed to stand on the bench for fifteen minutes, then 
moulded into loaves. 

In these test bakes the two loaves were carried through as one dough 
to facilitate handling and temperature control. The doughs were 
weighed and their temperature recorded at each punch. When taken 
to the bench for rounding, this dough was divided into two equal 
halves, and each half rounded up separately. In the first test bake 
the rounded doughs were moulded into loaves by hand, but in the 
second and third bakes uniformity of grain and texture was obtained 
by running them all through a Thomson machine moulder. 

The loaves were then panned in separate pans and placed in the 
proofing cabinet where they remained at a temperature of 32.5°C, in 
an atmosphere nearly saturated with moisture, until ready to go into 
the oven. The duration of the proof w*as usually 55 to 60 minutes, 
depending somewhat on the flour, and the height to wliich the doughs 
were allowed to rise before baking was that which was found by ex- 
perience to give the best appearing loaf with this type and size of pan. 

When the loaves were ready for the oven, the steam was turned on 
to furnish a moist heat and delay crusting, and allowed to remain on 
for the first three minutes the loaves were in the oven. The tem- 
perature of the oven was so adjusted that it registered 435° F at this 
point in the baking. Twenty to twenty-five minutes' baking was suf- 
ficient to produce a well baked-out loaf with a deep golden-brown 
colored crust. 

28 



The loa\-es were weighed directly out of the oven, again after one 
hour, and a third time at the end of eighteen hours. The volume of 
the loaves was taken at this time with the Central Scientific Com- 
pany's volume machine, using mustard seed, the volumes recorded 
having been repeatedly checked up and corrected by displacements in 
water. The loaves were scored and "placed" as to baking value, the 
resulting relative position being the result of independent judging by 
three experienced practical bakers. The numerical baking value 
assigned to each flour is the average of all the different scorings of 
three trial bakes, consisting of two loaves for each flour in each bake. 
The first series of baking tests was made in December, 1920. The 
baking on each loaf was repeated two or three times with slight varia- 
tion in fermentation time and absorption as indicated by the possible 
improvement of the loaf. The second test bake was made the middle 
of March, 1921 in order to properly classify some new samples just 
received. The order of baking value and relative strength of flours 
had not altered appreciably over that three months' period. The third 
baking test was completed in November, 1921. Only the data for 
this last series need be given here, since the relative position and score 
of the baked loaves remained the same over the six months' period. 
And though the flour had aged somewhat, as shown by the natural pH 
of the water extract, the slight change in "absorption" and slightly 
improved baking characteristics, the final bakings represent very well 
the best "strength" characteristics of each flour. 

Table VI is a record of the comparative baking tests; the final score 

being a summary of points according to the American Institute of 

Baking standard (1922). 

TABLE VI. 

Record of Comparative Baking Tests. 

Times Weights 

Fer- 

men- Proof- Dough 
Flour Absorb- tation ing at Dough Loaf Loaf Loaf Vol- 
Sample tion Period Period Mix M'lded Hot 1 hr. 18 hrs. umes Score 



No. 


% 


min. 


min. 


grams 


grams 


grams 


grams 


grams 


c c 




1009 


59 


255 


60 


564 


518 


457 


448 


435 


2160 


100 


1001 


58 


231 


60 


543 


539 




488 


460 


2010 


99 


1008 


60 


203 


53 


542 


535 


500 


492 


464 


2000 


97 


1002 


58 


225 


53 


532 


527 


491 




462 


1880 


95 


1012 


59 


225 


54 


537 


527 


489 




460 


1870 


91 


1006 


61 


195 


55 


541 


536 


494 


486 


471 


1735 


91 


1005 


59 


187 


65 


536 


531 


496 


484 


464 


1820 


90 


1010 


58 


165 


60 


531 


527 


491 


482 


468 


1760 


83 


1011 


56 


229 


51 


529 


524 


489 


479 


459 


1720 


76 


1003 


53 


186 


57 


520 




473 


464 


438 


1650 


63 


1013 


58 


224 


51 


528 


532 


496 


487 


468 


1630 


56 


1007 


65 


165 


46 


557 


552 


512 


500 


478 


1460 


46 


1004 


58 


192 


45 


549 




504 


498 


473 


1415 


35 


1014 


59 


206 


43 


540 




497 


490 


466 


1295 


32 



29 



Clarification. The chief objection to the application of autodiges- 
tion for the measurement of diastatic power has been due to the col- 
loidal character of the flour-water or malt-water suspensions. The 
longer the digestion continues the greater the degree of dispersion of 
these colloidal protein and dextrin products. Considerably difficulty 
has been encountered in obtaining a clear solution of sugars from 
enzymatic action and sufficiently free from those colloids which inter- 
fere with quantitative sugar determinations. Lead acetate as a clar- 
ifying agent for flour or malt solutions is exasperating in its slowness 
and poor results. It was found to be practically worthless for this 
work because of its failure to stop diastatic action. 

The previous inhibition of enzymatic activity by acid or alkali is 
not satisfactory because of the necessity of again neutralizing the 
solution before adding the lead reagent. 

The first obstacle to overcome was. therefore, that of clarification. 
The literature supplies numerous methods for investigation. Blish, 
(1918) made a study of protein precipitants and reported that 
"reagents ordinarily used for precipitating proteins, such as alcohol, 
acetic acid, trichlor-acetic acid, salts of heavy metals, colloidal iron, 
aluminum hydroxide cream, phosphotungstic acid, and tannic acid, 
are for various reasons unsatisfactory for removing gliadin from water 
extracts of flour." He recommended tenth normal copper sulfate and 
sodium hydroxide as the most efficient precipitant of protein nitro- 
gen. Phosphotungstic acid appeared to be the most serviceable for 
rapid work, and was the reagent used by Swanson and Calvin (1913) 
and by Thatcher and Koch (1914), for the clarification of their flour 
suspensions and extracts. The excessive cost, however, prohibits its 
use in quantity for control and service laboratories when other re- 
agents can be substituted. Folin and Wu (1919), developed a new 
protein precipitant, tunstic acid, which they applied to the precipita- 
tion of blood proteins. After the addition of one volume of ten per- 
cent sodium tungstate (NagWO^ .ZHgO.), to diluted blood serum 
they added, with shaking, one volume of two-thirds normal sulfuric 
acid. The resulting precipitate was in such form that it could be 
easily centrifuged and filtered. The acid is intended to set free the 
whole of the tungstic acid and to neutralize the carbonates usually 
present in the commercial tungstate, with about ten percent in exces.-.. 
Because of the efficiency and ease of application, combined with a 
greatly reduced expense, it would seem that this reagent deserves a 
wider application in the clarification of colloidal protein suspensions 
than it enjoys at present. A few trials on flour suspensions, extracts, 
and malt syrups gave promise of its being better suited to these than 
any other method yet employed. 

30 



Preliminary experiments showed that 2. cc of a 15% sodium tung- 
state solution w;ere sufficient for the soluble proteins in 5 grams o' 
flour, or 3. cc for 10 grams. These preliminary trials did not always 
result in a good clarification even though the equivalent amounts of 
2/3 N. H2SO4, as suggested by Folin and Wu, were employed. Some- 
times the supertant liquid became clear almost immediately, the 
flocculated proteins settling out rapidly, and again the cloudiness per- 
sisted after a half hour's centrifuging. The reason was not far to 
seek. The addition of the sodium tungstate to a suspension of sound 
flour in water produces an alkaline reaction. The complete precipita- 
tion of the proteins from a colloidal suspension depends upon their 
adsorption to the tungstate ion and the subsequent precipitation of the 
coagulated aggregate by throwing the hydrogen ion concentration 
over sufficiently far to the acid side of the isolectric point. This was 
accomplished by Folin and Wu through the addition of 2/3 N.H.^SO^ 
in quantity which neutralized the combined alkalinity of the tungstate 
and blood serum with a slight excess; but in the case of flour extracts 
the higher buffer value * of the phosphate and other salts present re- 
quires a much larger excess of acid to produce the necessary hydro- 
gen ion concentration. A number of clarifications were obtained in 
which the supernatant liquid became as clear as water after a few 
minutes centrifuging. The hydrogen ion concentration of these solu- 
tions was determined electrometrically and they were found to have 
values in terms of pH ranging from 2.117 to 1.337. ** The slightly 
cloudy liquids from the unsatisfactory clarifications all showed pH 
values of 2.67 or above. 

These and subsequent results confirm the fact that the success of 
the sodium tungstate clarification depends upon proper acidification 
of the NagWO^ - protein suspension to a hydrogen ion concentration 
of I.XIO"- or more, corresponding to a pH of 2.0 or less. There is 
no danger of precipitating the colloidal tungstic acid hydrate even with 
a much larger concentration of acid. Instead of the 2/3 HgSO^ of 
Folin and Wu, or the 1. N. acid which was first used in these experi- 



*(The term "buffer" was introduced by Fernbach and Hubert (Comptes 
rend. Acad. Sci. 131,293 (1900). It was repeatedly used by Sorenson 
(Comptes rend, du Lab. de Carls. 8,53 (1909), (Ergebnisse der Physiologie 12,- 
523 (1912), by Henderson (Ergebnisse der Physiologie 8,254 (1909) and by 
Jenny Hcnipel (Comptes rend, du Lab. dc Carls. 13,1 (1917) to designate the 
effect of various salts in the media which dissociate upon the addition of acids 
or alkalies and therefore "use up" different amounts of the titrating reagent 
before significant changes are brought about in the hydrogen ion concentration 
of the media. The importance of these buffer salts in wheat flours has been 
emphasized by Bailey and Peterson (Jour. Ind. Eng. Chcm. 13,916 (1921). 

**(A11 electrometric determinations were made with the Leeds and 
Northrup Type K Potentiometer, using the Bailey (1920) hydrogen electrode. 
The pH values corresponding to the millivolt readings were taken from the 
Schinidt and Hoagland tables.) 

31 



merits, concentrated sulphuric acid added from a micro-pipette ap- 
pears to be more efficient in precipitating the protein-tungstate. The 
precaution of adding the acid slowly, drop by drop, with shaking of 
the solution must be observed, otherwise a local concentration will 
produce a precipitation of flocks of colloidal tungstic acid, and with 
further danger of decomposing some of the carbohydrates. Thymol 
Blue (Clark 1920) with its acid range at pH 2.+ serves as a very con- 
venient indicator for the first few trials with any new sample, since it 
is necessary to add only two or three drops of the concentrated acid 
in excess of the pink color to produce the proper acidity for com- 
plete precipitation. After a few trials uniform results were always 
obtained by measuring the acid from a 1. cc micropipette or counting 
the number of drops required to produce the proper color by Thymol 
Blue. 

Because of their higher buffer value malt flour suspensions are 
found to require a slightly larger amount of acid. In this connection 
a difficulty was later encountered in the clarification of suspensions 
to which considerable quantities of acid had already been added. The 
same principle of colloidal protein precipitation applies here as well, 
and the clarification is satisfactory if the suspension is first neutral- 
ized by means of a few drops of strong NaOH. The Thymol Blue, 
alkaline range, blue color (pH 8.0 to 9.6) likewise serves for this point, 
and though the hydroxyl ion concentration does not need to be carried 
so far, it does no harm as the subsequent addition of acid brings it 
back immediately to a pH of 2.0 or less. 

The suspensions to be clarified in this series were always centrifuged 
for two or three minutes to save time, and more especially to form a 
compact mass of flour solids in the bottom of the centrifuge tube from 
which the clear supernatant liquid could be poured or pipetted without 
stirring up any of the material which had been thrown down. But if 
a centrifuge is not available the starch and precipitated proteins settle 
out clear in about five minutes, and if desired the supernatant liquid 
can be rapidly filtered through a fine quantitative filter paper. This 
is especially true of the clarified dilute solutions of malt extracts in- 
tended for sugar determinations. As will be shown later, however, 
the sodium tungstate clarification followed by a few minutes centri- 
fuging effects the elimination of all filtrations, which heretofore have 
required hours, and which because of the errors thus introduced have 
been the stumbling block in many investigations of this nature. 

The amount of soluble nitrogen remaining in solution was deter- 
mined by the Kjeldahl method before and after clarification, using 
different amounts of tungstate and varying acidities. The residual 
nitrogen appears to reach a fairly constant minimum for the flour used 

32 



under the conditions of clarification described above. Different ma- 
terials show different content of solublie amino nitrogen and ammonia 
nitrogen which is not removed by the tungstate procedure, but which 
shows no vitiating effect on the reducing sugar determinations. The 
results of the above discussion are summarized in table VII, which 
shows the nitrogen remaining in solution after treatment with sodium 
tungstate. 



TABLE VII. 

The efficiency of the Sodium Tungstate reagent as a clarifying 

agent for flour suspensions at various 

concentrations and acidities. 













Nitrogen 












Remain- 




Flour 
grams 


Final 

Volume of 

Clarified 

Solution 


Na.WO 
15% 


Acid Added 


pHof 
resulting 
Solution 


ing in 

100 
cc's of Remarks on 
Solution Clarification 


5 


cc. 
100 


cc. 

4 


S.ccT-jN H.SU^ 


3.88 


grams 
.0034 


Fair 


5 


100 


2 


2.5 cc N/1 H=SO« 
10 drops in excess 
by Thymol Blue* 


2.603 


.0021 


Excellent 


5 


100 


3 


Acid by Methyl 
Orange 


5.04 


.0129 


Poor; very 
cloudy 


5 


100 


2 


10 drops in excess 
by M. O. 


3.446 


.0027 


Good 


•) 


100 


2 


Acid by Thymol 
Blue* 


2.536 


.0021 


Very good 


10 


200 


3 


4. cc N/1 H.SO. 


2.117 


.0026 


Excellent 


10 


200 


3 


.4 cc Cone. H.SO« 


1.468 


.0021 


Excellent after 
1 hr. digestion 


10 


200 


3 


.4 cc Cone. H2SO4 


1.457 


.0021 


Excellent after 
3 hrs. digestion 


10 


200 





0. cc 


5.778 


.0241 


No clarification 



10 



200 



0. cc 



after 1 hour di- 
gestion. 

5.596 .0379 ^o clarification 
after 3 hours 
digestion 



*(7. drop.s Thymol P.lue Indicator). 



33 



Reducing Sugars. Several methods present themselves for the de- 
termination of reducing sugars in the clarified solutions from flour, 
or malt enzyme digestions, and different ones have found favor with " 
the different workers on the products of diastatic action. Swanson 
and Calvin (1913) Thatcher and Koch (1914), and Thatcher and Ken- 
nedy (1917), applied the iodine titration method to the residual copper, 
after the Fehlings reduction, basing their work on the articles pub- 
lished by A. W. Peters (1912). Spoehr (1919) modified the details of 
the same method for application to small amounts of sugar-containing 
juices from cacti. He found that his solutions contained other sub- 
stances than sugar which contaminated the weight of reduced cuprous 
oxide and so made it necessary to determine the unreduced copper. 
Harter (1921) followed the Clark (1918) modification of the Scales 
(1915) procedure to measure the reducing sugars produced by the 
diastase of Rhizopus tritici. The method of Bertrand (1910), which 
determines the amount of copper reduced by the sugars, has been but 
little used in this sort of work. The Bertrand titration method fur- 
nishes a convenient way of checking the weights of the precipitated 
cuprous oxide, and was so used in this laboratory to determine the ac- 
curacy of the crucible weights as compared to the actual copper re- 
duced. In this way it was found that the supernatant liquids from the 
tungstate clarification as carried out in the present investigation gave 
accurate and concordant results by weighing the filtered cuprous oxide 
precipitate, and the difficulty in contamination of the reduced copper 
by non-sugars, as recorded by Spoehr, was therefore not encountered. 

Because of the conditions under which this work was done, it was 
found more convenient to use the prepared gooch crucibles with asbes- 
tos mats for filtering the reduced copper than to apply the iodine 
method of titration, with its consequent necessity for the preparation 
of solutions. The total time required for the preparation and weigh- 
ing of the gooch crucibles is hardly greater than that for the titration 
procedure. Furthermore, the excellent contribution of Shaffer and 
Hartman (1921) has shown that the iodometric titration method for 
residual copper as previously carried out is not without its possibili- 
ties of serious error. On the other hand Ouisumbing and Thomas 
(1921) in their recently published article on the reduction of Fehling 
solution by different sugars, point out several possible sources of er- 
ror in the reducing sugar determinations according to the official Mun- 
son-Walker procedure. Unfortunately these two articles did not ap- 
pear until the work reported here was well along toward completion, 
and it was believed best to complete the determinations using the same 
comparative procedure ; consequently all reducing sugar determina- 
tions reported in the experimental part of this paper were made by 

34 



the Munson-Walker method, (A. O. A. C. Methods of Analysis, re- 
vised, 1919. Section VII.) A battery of forty No. porcelain gooch 
crucibles were used, and the cuprous oxide resulting from the Feh- 
lings reduction was filtered onto thick asbestos mats, washed, dried 
and weighed. The corresponding weights of maltose were taken from 
the Munson-Walker tables. The crucibles were fitted with mats of 
properly prepared washed and ignited asbestos, at least 1 cm. in thick- 
ness, and washed into place by whirling with a stream of hot water 
from a wash bottle. They were then washed with 95% alcohol and 
dried at 100° for 1 hour. The weights of the crucibles so prepared 
remained constant in the dessicator for periods of weeks. 

The Fehling solutions used showed no auto-reduction in the blanks. 
The only standardization necessary was to run a few blank determina- 
tions in order to apply a correction for each new lot of asbestos pre- 
pared. The' 400 cc Pyrex beakers used for Fehling solution reduc- 
tions were of approximately uniform thickness, the watch glasses used 
for covers fitting rather closely. The flame was so adjusted that the 
different samples started boiling usually within five seconds of the four 
minute period stipulated. The chief source of error was found to be 
in the loss of the weight show'n by different lots of prepared asbestos 
upon pouring the hot Fehling solution through the mats. The maxi- 
mum variation seldom went beyond 1 milligram and with triplicate 
determinations the weights of Cuprous oxide usually checked within 
.2 to .3 milligrams of the average. It would appear highly desirable 
for this sort of work to combine the method of Shaffer and Hartmann, 
and of Quisumbing and Thomas into a standard method in which the 
sources of uncontrolled error are reduced to a minimum. Also the ap- 
plication of sodium tungstate as a clarifying agent for protein-contair- 
ing solutions could be profitably studied in its connection with such 
a method. 

Effect of Clarifying Agent on Sugar Determinations. The next 
point requiring investigation was the eft'ect, if any, of various amounts 
of the sodium tungstate, as used for clarification, on the determina- 
tion of reducing sugars by Fehling solution. Preliminary trials on 
flour-water suspensions with and without added dextro -e indicated 
that the addition of the sodium tungstate in excess for clarification did 
not affect the Fehling's reduction. To confirm this point and to see 
what results could be obtained from unclarified solutions, the follow- 
ing method was used : five grams of a flour showing considerable dia- 
static activity were weighed into 250 cc beakers, 50 cc of water at 27°C 
were added, and the mixture thoroughly stirred. In some cases 40 cc of 
H.,0 and 10 cc of a 0.5% dextrose solution were added in place of the 
water alone. These were allowed to digest for one hour at 27°C, stir- 

35 



ring at intervals. Some of the samples were then clarified by Na,,WO^, 
using Thymol Blue as indicator. In the other samples the enzymic 
activity was inhibited by 5. cc of 0.2N NaOH. After clarification, or 
inhibition of the enzymic action, the solution was diluted to 100 cc in 
a volumetric flask, poured into a 100 cc centrifuge tube and whirled 
ten minutes. 50 cc of the clear supernatant liquid were thern pipetted 
out into a 400 cc beaker for determination of sugars by the Munson- 
Walker method. 

The results are compiled in table VIII under three groupings, 
samples numbered 1 to 4 include the blanks, 5 to 7 the flour with no 
added dextrose, and numbers 8 to 13 the flour samples with a known 
weight of dextrose added. 

TABLE VIII. 

The determination of reducing sugars in solutions with and 
without clarification by sodium tungstate. 









1; 


':>% Na: 


:WO 


4 


Average 


Expressed 


Sample 






Dextrose 


For 




Cu.O 


Weight 


as Dextrose 


Number 


Flour 


Added CI 


arification 


Weighed 


of Cu.O 


Recovered 




gra: 


ms 


milligrams 


cc. 




grams 


grams 


milligrams 


1. 


0. 




0. 


0. 




-.0005 
-.0006 


-.0005 


none 


2. 


0. 




.SO. 


0. 




.1146 
.1150 


.1148 


49.7 


3. 


0. 




0. 


2. 




-.0004 
-.0002 
-.0006 


-.0004 


none 


4. 


0. 




.SO. 


2. 




.1138 
.1156 
.1140 
.1145 


.1145 


49.6 


5. 


5. 




0. 


0. 




.0800 
.0788 
.0795 


.0794 


34.0 


6. 


5. 




0. 


2. 




.0795 
.0782 
.0795 
.0800 


.0793 


34.0 


7. 


5. 




0. 


2 




.01665* 


.01665 


6.8 


8. 


5. 




.SO. 


0. 




.2008 
.2004 


.2006 


89.0 


9. 


5. 




50. 


1. 




.1985 
.1987 


.1986 


88.6 


10. 


5. 




50. 


2. 




.1997 
.2004 
.1963 
.1963 


.1982 


88.4 


11. 


5. 




50. 


5. 




.2013 


.2013 


89.4 


12. 


5. 




50. 


10. 




.2027 


.2027 


90.4 


13. 


5. 




50. 


2. 




.1975 
.1984 


.1980** 


88.0 


*Average 


of 5 determinations. 










** (Filtered) 















36 



The values recorded as sample number 7 are the average found for 
five determinations of the natural reducing value of the flour, the dias- 
tase having been inhibited by preliminar}^ tungstate and acid treat- 
ment. 

The determinations recorded in Table VIII v^^ere obtained under 
conditions of poor temperature control. The De Khotinsky electric- 
ally heated constant temperature water bath with electrostatic control 
which was used for all subsequent work had not yet been installed, 
and these digestions were made at 27° C in an incubator. The tem- 
perature fluctuated rather widely (around .5 degree), and the conse- 
quent variation in diastatic activity was to have been expected. This 
series was not repeated as such, because later data obtained in con- 
nection with other experiments has shown that the conclusions as in- 
dicated by Table VIII are valid, namely; (1) The use of 15% sodium 
tungstate in quantities up to 5 cc, and sulfuric acid for clarification of 
flour suspensions, neither interferes with, nor affects the determina- 
tion of reducing sugars in the clarified solution by the Munson-Walker 
method. (2) It is not absolutely necessary to clarify the supernat- 
ant liquid obtained from the centrifuging of a 5% flour and water sus- 
pension. This should be further qualified by notes on the appearance 
of the Fehling's reduction. It was shown in every case that clarifica- 
tion was an advantage in the reduction of Fehling's solution, espec- 
ially in the case of cloudy solutions. If unclarified, the boiling Feh- 
ling solution foamed' badly, and the formation of CugO appeared to be 
hastened by the coagulated protein, resulting in a dark and somewhat 
muddy looking precipitate which filtered badly, and showed a ten- 
dency to adhere to the surface of the beaker, (3) The use of the 
sodium tungstate clarifying reagent renders the solution clear and 
protein-free, and when centrifuged to throw down suspended matter 
it eliminates all necessity for filtration. Further proof that filtration 
of the clarified centrifugate is entirely unnecessary, is evident from 
data obtained in other experiments, at different times and for difl"er- 
ent purposes, but using the same flour sample and method. The ob- 
ject in view when these samples were run bore no intentional relation 
to conclusion (3), but the results substantiate those obtained in table 
VIII. This data is collected and tabulated in Table IX. 

TABLE IX. 
The determination of reducing sugars in the centrifugate from the 
sodium tungstate clarification before and after filtering. 
Sample Grams CU2O 

1 Filtered .12591 

2 Filtered .1259 I- .1256 Average 

3 Filtered .12S2J 

4 Unfiltered .12601 

5 Unfiltered .1251 > .1255 Average 

6 Unfiltered .1256J 

37 



On the other hand, a \ ei}- few trials are sufficient to convince one 
that filtration of the unclarified solution from a flour-water suspen- 
sion is a most unsatisfactory procedure and should be expected to 
give inaccurate results for reducing sugars. 

A further careful scrutiny of the data in table VIII brought out a 
peculiarity in the determinations which has not yet been satisfactorily 
explained. Comparing samples Nos. 5, 6 and 7, and Nos. 1, 4 and 7, 
it appears that there is an increase of reducing sugars as determined 
by weighing the CuoO over and above that added in the form of dex- 
trose. This question, like several others which were brought out in 
the course of this series of investigations, is recorded here with what- 
ever explanation appears possible from the data at hand, with the 
hope of enlisting the interest and experimental efforts of other work- 
ers. The most obvious explanation is that the natural reducing value 
of the flour extract is due (probably) to dextrose, while the increase 
of reducing power after one hour of diastatic activity is due to mal- 
tose. In table VIII the weights of Cu.O as recorded are produced in 
some instances by dextrose alone (samples numbered 1 to 7 inclusive), 
and in others by maltose alone or by both dextrose and maltose 
(samples 8 to 13 inclusive). 

TABLE X. 

The relation of actual to calculated values for the reducing power 

of dextrose added to autolytic digestion of diastatic flour. 

(Data taken from Table VII). 

A B 

In terjns of In terniis of Dextrose 
Cuprous Oxide and Maltose 
Without added dextrose Milligrams Milligrams 
Total reduction after 1 hour's diastasis (sam- 
ples Nos. 5 and 6) 79.35 60.88 = Maltose 

Blank (natural reducing power of flour (sam- 
ple No. 7) 16.65 6.86 = Dextrose 

Difference due to diastasis 62.70 54.02 = Maltose 

With added Dextrose 

Dextrose added (sample Nos. 2 and 4) 114.58 49.83 = Dextrose 

Blank (natural reducing power of flour (sam- 
ple No. 7) 16.65 6.86 = Dextrose 

Total, due to Dextrose 131.23 56.69 = Dextrose 

Total reduction after 1. hour diastasis (sam- 
ples Nos. 8 to 10 inclusively) 199.51 

Total reduction due to Dextrose 131.23 56.69 = Dextrose 

Difference due to diastasis 68.28 52.12 = Maltose 

108.81 = Total 

reducing 
sugar 

Value found 62.70 108.81 

Value calculated 68.28 110.77 

Increase 5.58 mg. 2.96 mg. 

38 



In Table X the data has been rearranged to show that the increased 
weight of cuprous oxide, in the range of values between 63 and 131 
milligrams, is equivalent to a positive error of 5.58 milligrams of 
dextrose, or an increase of nearly 9% over the calculated value. Since 
the average error of the method, due to slight differences in tempera- 
ture, volumes and weights is about 0.8 milligram of Cu^O, the above 
variation is more likely due to other causes. When the data is recal- 
culated into terms of dextrose and maltose respectively, as shown 
under Part B of Table X, the error is reduced somewhat, due to the 
smaller numerical value of the corresponding reducing sugars. This 
method of calculation gives a difference of approximately 3. milli- 
grams of reducing sugars, or about 5. per cent. Differences of similar 
magnitude, and positive in sign, have been obtained in subsequent 
work. 

Adsorption by Starch. The next explanation to suggest itself is a 

displacement concentration due to the volume occupied by the 5. 

grams of flour in the 100 cc flask. It will be shown that this accounts 

for only part of the error. The hydrated colloids of the flour, e. g. 

starch and gluten, might be expected to adsorb some of the sugars, 

thus reducing the concentration of the supernatant liquid. Thus 

there would exist a ratio between the reduction in the concentration 

of sugars due to adsorption, and the increase of concentration due to 

volume displaced by the flour. The difference then in result between 

the dextrose recovered, and the dextrose added to the flour suspension 

plus the reducing value of flour-water suspension, would be resultant 

Displacement 

of this ratio of—rr- ^— ^ concentration. 

Absorption 

We should also expect these colloids to be sensitive to changes of 
electrolytes or of hydrogen ion concentration, yet the results of sever- 
al trials did not show sufficient adsorption of dextrose by flour colloids 
in excess water at a pH of () or less to be recognized by the methods 
employed. On the other hand, the errors were always in the opposite 
direction, that is, there was always an increase in sugars recovered 
over that expected. 

Samples of both wheat flour and wheat starch were weighed into 
accurately graduated flasks, the flasks fiilled to the mark with distilled 
water, and allowed to stand either one hour or 24 hours, before being 
brought again to volume and weighed. From these weights it was 
calculated that .5601 cc of water is displaced per gram of flour, and 
.7086 cc per gram of starch. Using these values to calculate the dis- 
placement concentration, the total increase in weight of the CujO, 
due to increased concentration, should be approximately 2.2 milli- 
grams. This is less than half the difference actually obtained. For 

39 



want of a better explanation the remaining error might be laid to the 
increased reduction of the Fehling solution by the addition of dextrose 
to the maltose in solution. Such a probability is suggested by the 
recently published work of Quisumbing and Thomas (1921). 

Preparation of Wheat Starch. This increased recovery of reducing 
sugars by Fehling's reduction is likewise shown by some of the data 
obtained on starch. To obtain some idea of the behavior of starch 
in flour under the conditions of these experiments, several samples of 
wheat starch were washed out of flour with running water, the gluten 
being held back by manipulating over a No. 10 flour silk gauze. 
Repeated washing, decantation, and differential centrifuging failed 
to remove all the protein matter from the starch, which still showed 
some slight diastatic activity after driying. Other samples were 
then prepared and the proteins removed by dispersion and wash- 
ing with very dilute NaOH. Too strong soda solution gelatinizes the 
starch, rupturing the granules and even though a concentrated alcohol 
treatment retrogrades it back to insoluble starch (Herzfeld and Kling- 
er, 1921.), the physical and chemical character of the material has been 
changed, and it is no longer comparable to the raw wheat starch of 
the flour. After the proteins and NaOH have been washed out with 
distilled water, the starch is carefully centrifuged, and only the center 
portion of the column deposited in the liottom of the centrifuge tube is 
taken. This is repeatedly washed with cold distilled water containing 
a few drops of HCl per liter until no more acid is removed from the 
solution. The material is then filtered on a Buchner funnel, washed 
w ith re-distilled neutral alcohol, and dried in a vacuum at 105° for five 
hours. After cooling in a dessicator, the samples were allowed to re- 
absorb moisture from the atmosphere for several days, and bottled. 
This prepared starch could then be weighed out in the open balance 
without errors due to rapid changes in weight by moisture absorption. 
Its moisture content was determined. These starches showed no dias- 
tatic activity nor reducing sugars. 

Samples of this air dried starch were weighed into 250 cc beakers, 
and 50 cc of water added to each at 27° centrigrade. Ten cubic centi- 
meters of 1% dextrose were then pipetted in, well stirred, and allowed 
to remain at 27°. At the end of exactly one hour they were diluted to 
100 cc in a calibrated volumetric flask, centrifuged clear, and 50 cc of 
this clear supernatant liquid taken for determination of reducing 
sugars. To determine whether the change of acidity by the Na^WO^ 
clarification procedure would affect the adsorption of sugars by starch, 
and to check up on the eft'ect of clarification upon Fehling's solution 
reduction (table VIII. page 33), alternate samples were clarified at the 
beginning of the digestions. In other words 3. cc of Na^WO^ (l57o) 

40 



and .4 cc cone. HoSO^ were added immediately after the 10 cc of dex- 
trose solution. The results are shown in table XL 

TABLE XI. 

The recovery of reducing sugars after addition to starch in water. 













OUgcll .1 


After Correction 


umber 


Starch 




Sugars 






For Volume 




as Substrate 


pH 


Added 


Bv Ai 


iialysis 


Displacement 




Grams 




Milligrams 
Dextrose 


Mill 


i grams 


Milligrams 


1. 


3.0 


5.092 


48.4 




50.4 


49.2 


2. 


3.0 


1.790 


48.4 




49.9 


48.8 


3. 


4.0 


5.000 


24.2 




25.2 


24.5 


4. 


4.0 


1.618 


24.2 




24.7 


24.0 


5. 


5.0 


5.581 


48.4 




50.4 


48.5 


6. 


5.0 


1.932 


48.4 




50.5 


48.7 


7. 


7.0 


5.466 


48.4 




53.5 


50.7 


8. 


7.0 


1.900 


48.4 
Maltose 




51.5 


48.6 


9. 


4.0 


4.600 


40.9 




43.8 


42.4 


10. 


4.0 


1.615 


40.9 




42.8 


41.5 


11. 


4.0 


1.900 


61.4 




64.9 


63.0 



The last column of figures in table XI shows the calculated weights 
of CuoO after correcting for the concentration displacement by the 
starch in 100 cc volume. The corrected values fall almost within the 
limit of experimental error with the exception of samples numbers 1, 
7, 9 and 11. The good agreement of the pairs of results also confirms 
a conclusion of table VIII, namely, that the Na^WO^ clarification has 
no effect on Fehling solution reduction. It is also evident from the 
data given in Table XI that there is no adsorption of reducing sugars 
by the starch. 

Inhibition of Enzyme Activity. Much has been published on the 
effect of the so-called catalytic poisons on various enzymes. The re- 
sults are so numerous and the conclusions so contradictory that it 
would serve no purpose to review^ them at length. It has been shown 
that each enzyme poison, whether it be an antiseptic, or heavy metal 
salt, must be conisdered separately and in its relation to the particular 
enzyme in question. In general, the salts of the heavy metals, antisep- 
tics, and in fact most of the so-called catalytic poisons, have but little 
inhibiting effect on the activity of diastase under normal conditions 
of pH and salt concentration except as they are able to precipitate the 
enzymic carrying protein or to inflvience the pH of the medium. The 
work of Sherman and Caldwell (1921), has further emphasized the 
protective action of the amino acids on the enzymes in the presence of 
catalytic poisons. Many of those who have investigated diastatic 
activity have recorded the use of NaOH in varying concentrations to 
stop enzymic action. Others have employed low temperatures. The 

41 



effect of continually increasing amounts of alkali on diastatic diges- 
tions is shown later in Table XYY and Fig. 3. The concentration of 
alkali required for complete inhibition of flour diastase is considerably 
higher than that generally recomended, and there would seem to be 
serious danger of destroying some of the sugars present by the addi- 
tion of such hydroxyl ion concentration, as pointed out by Neff", 
(Armstrong, 1919.)- Furthermore, the increased dispersion and solu- 
tion of proteins by the added NaOH is not desirable. The unsatisfac- 
tory results obtained in preliminary experiments, whenever NaOH 
was used to stop diastatic activity, or lead acetate for clarification of 
flour suspensions, in contradistinction to the uniformly satisfactory 
results by the use of sodium tungstate and sulphuric acid, lead to the 
experiments which are summarized in Table XII. 

Ten grams of flour were weighed out into 300 cc flasks and 100 cc 
of distilled water added by pipette, with continual shaking to get the 
flour thoroughly stirred throughout the liciuid. The various mater- 
ials to be used as inhibiting agents were then added and the whole 
allowed to remain in the water bath for the lengths of time as desig- 
nated in the table, (shaking up at fifteen minute intervals). Sample 
number 1 was clarified by the tungstate reagent at the end of the first 
hour. Sample number 2 was clarified at once, then allowed to stand 
one hour before determining the reducing sugars. Sample number 3 
was clarified in the same manner as number tw'o, except that it was 
allowed to stand two hours before determining the reducing sugar.i. 
Sample four was tried with only 3. cc of sodium tungstate, on.itting 
the precipitation by sulfuric acid until the end of the hour. Samples 
five and six show the effect of two different concentrations of alkali 
previously recommended in the literature for the inhibition of dias- 
tatic activity. Samples seven and eight w^ere treated with 2. cc of 
basic, and 2. cc of neutral lead acetate, respectively, and allowed to 
stand one hour before filtration and removal of the lead for sugar de- 
terminations. They were then clarified and treated acconb'ng to the 
official A. O. A. C. procedure. Sample number 7 was cloudy and re- 
quired twelve hours for filtration, while sample eight filtered much 
more rapidly. Samples nine and ten were cooled to zero degrees by 
an ice and salt bath. In samples 1, 5, 6, 9, and 10, the diastatic activ- 
ities were stopped by the sodium tungstate clarification in the manner 
described above, while sample 4 required the addition of .4 cc concen- 
trated H0SO4. After clarification the samples were all made up to a 
volume of 200 cc, centrifuged, and 50 cc aliquots taken for reducing 
sugar determinations. The results are expressed in table XII in terms 
of cuprous oxide corresponding to the reducing sugars from 50. cc of 
the clarified solution (2.5 grams flour.) 

42 



TABLE XII. 
The effect of different inhibiting agents on the diastase of flour. 



Sample No. 


Inhibiting 
Agent 


Time and 
Temperature 


Weight CujO 
per 50. cc's 
of Solution 
(2.5 g. flour) 


pH of 
Digestion 


1. 


none 


1 hr. at IT 


.0833 




2. 


(Na=W04 
+ H2SO4) 


1 hr. at 27° 


.0140 


below 2.0 


3. 


(NaaWO. 
+ H,SO,) 


2 hrs. at 27" 


.0141 


below 2.0 


4. 


(Na2W04 
only) 


1 hr. at 27° 


.0491 


7.241 


5. 


2. ccO.lN. 
NaOH) 


1 hr. at 27° 


.0316 




6. 


(5cc O.IN. 
(NaOH) 


1 hr. at 21° 


.0169 


9.1336 


7. 


PbAc (Neut.) 


1 hr. at 21° 


.1034 




8. 


PbAc (Basic) 


1 hr. at 27° 


.0066 




9. 


Ice 


1 hr. at 0° 


.0287 


5.981 (21. °C) 


10. 


Ice 


3 hrs. at 0° 


.0388 


5.981 (21. °C) 



Confirmatory results similar to these and leading to the same con- 
clusions have been obtained on two other flours (Our No. 1 and 1003). 

Samples numbers 2 and 3 of Table XII, as examples typical of many 
similar determinations, show conclusively that the use of sodium tung- 
state and sulfuric acid, with a final pH of between 1.5 and 2, effectual- 
ly stop diastatic activity. This is accomplished both by complete pre- 
cipitation of the enzymic active protein and by effecting a hydrogen- 
ion concentration which itself inhibits diastatic activit3\ The concen- 
tration of acid is not sufficient to further hydrolyse the disacharides or 
dextrins, even after several hours standing. This had been previously 
pointed out by Swanson and Calvin, who used a final concentration of 
.02N H2SO4, which corresponds to a pH of somewhere between 2 and 
2.5. This fact allows an accurate measurement of the reducing sug- 
ars by gravimetric Fehling's determination at the operator's conven- 
ience. 

Samples 5 and 6, Table XII, indicate that NaOfI is not a reliable 
inhibiting agent to u.se for diastatic ])reparations where the quantity 
of buffer is as high as that found in flours or malts, unless a large con- 
centration is used. There is also the probable danger of destroying 

43 



part of the reducing sugars present if too high a concentration of al- 
kali be used. 

The use of lead acetate, (table XII, numbers 7 and 8), gives variable 
results depending upon the concentration of the reagent, the concen- 
tration of the material used to de-lead the solution, and the time con- 
sumed in the operations of filtration. 

In point of time alone the sodium tungstate procedure requires only 
about five minutes, with the elimination of all filtration, which is a 
great advantage in enzyme work, while the clarified solution can be 
allowed to stand for several hours, or until all of the samples are 
ready for the Fehling reduction. 

Samples numbers 9 and 10 (table XII), clearly indicate, that the 
temperature of an ice bath will not stop diastatic activity in solutions 
where the diastase is protected by buffers naturally occurring in the 
plant medium. In the two trials the temperature within the flasks 
was held at zero by means of salt and ice, and they had to be stirred nt 
short intervals to prevent ice crystals forming on the inside surface of 
the flasks. Other samples with temperatures around 5°C showed very 
nearly the same results. Samples 2 and 3 (Table XII), in conjunction 
with results of nearly a hundred other similar determinations, serve to 
show this "blank" gives very constant and duplicable results, and af- 
fords a measure of the reducing sugars present in the flour. While 
this reduction of Fehling solution is due to dextrose, and the total 
reduction by autolytic digestion of the flour-water suspension is a re- 
sult of added maltose produced by diastatic action, the difference in 
the weight of Cu.,0 produced give a reliable measure of the maltose. 
This procedure for the determination of original reducing sugars in 
the sample should be directly comparable to the values obtained from 
the official alcohol extraction method. Likewise, it should be pos- 
sible to substitute the sodium tungstate clarification for the lead 
acetate procedure in the alcohol-extract of sugars from flour and malt 
products. Preliminary experiments on the application of this new 
procedure for both reducing and total sugars have indicated its suc- 
cessful application. Collaborative w'ork with the Department of Ag- 
riculture, Bureau of Chemistry, is now being conducted on this prob- 
lem and the results will be reported at a later date. 

Effect of Concentration. In general, investigators of enzymic ac- 
tivities have found it necessary to take the concentration effect into 
consideration before establishing their conditions for observations and 
measurements on enzyme preparations. This concentration effect is 
especially important when working with extracted and purified en- 
zyme preparations. In the preliminary discvission of factors which 
may limit the activity of diastase in one way or another two such fac- 

44 



tors were considered, namely, amyloclastic power, and variable resist- 
ance of different starches to amyloclastic action. 

As a third limiting factor the concentration effect on diastatic ac- 
tion should receive attention because of its peculiar relation in this 
series of measurements. Before diastatic enzymes can act to best ad- 
vantage there must be sufficient water present. In the germinating 
seed this is assured and controlled: through imbibition of water by the 
colloidal system. In a dough batch the concentration is likewise con- 
trolled by the amount of water the flour colloids can imbibe and still 
retain sufficient elasticity and tenacity to handle w'ell in baking prac- 
tice. (Ostwald 1919, and Luers and Ostwald 1919-1921). This de- 
gree of concentration varies considerably with dift'erent flours and in 
the hands of different bakers, within certain limits. When compared 
to the long period of time required for germination, the short fermen- 
tation of a dough should show concentrations which are more or less 
comparable. 

To determine what effect concentration w,'ould have on the activity 
of diastase as measured by the methods employed in this report, two 
types of experiments were carried out. In one, attempts were made 
to measure the reducing sugars produced in a dough fermentation 
containing percentages of water comparable to the normal absorption 
as obtaining in bake shop practice, and in the other, to determine the 
concentration effect in dilutions such as are used in the method fi- 
nally chosen for measurements of comparative diastatic powers in the 
standard flour samples. 

In Table XIII the first three values given are those for diastatic 
activity in the doughs, and were obtained as follows : 50 grams of 
flour were weighed into 250 cc beakers and allowed to come to a tem- 
perature of 27°C. Distilled water at 27°C was added from a burette 
in volumes ranging from 27.5 cc to 50 cc, or in other words in amounts 
corresponding to absorptions of 55 to 100%. The flour and vvater was 
thoroughly doughed up by means of a stiff spatula and the stiffer 
doughs were kneaded a minute in the fingers. This dough was 
pressed into the bottom of the beaker, covered, and placed in the water 
bath of 27° for one hour. At the end of that time the dough was 
weighed and one-fifth of the total weight, corresponding to ten grams 
of flour, was taken for determination of sugars. This aliquot sam- 
ple of dough was transferred to a 250 cc bottle, 100 cc of water 
were added, and several drops of 50% NaOH to aid in dispersing the 
gluten and slow up enzymatic activity. The bottle was quickly stop- 
pered and shaken for a few minutes to completely disperse the gluten 
and dissolve out the sugars. The contents of the bottle were then 
rinsed into a 200 cc volumetric flask and clarified by sodium tungstatc 

45 



and sulfuric acid as described above. From the centrifuged super- 
natant solution 50 cc aliquots were pipetted out for reducing sugar 
determinations. The values for the other four concentrations as re- 
corded in the same table were obtained by varying the ratios of the 
flour-water suspensions from 1 : 2.5 up to 1 : 40. and allowing the mix- 
ture to digest autolytically for one hour at 27°. The diastase in 
these samples was then stopped by the regular sodium tungstate pro- 
cedure, made up to volume, and the reducing sugars determined in 
the centrifugate in the same manner as for the other three samples. 

TABLE XIII. 

The effect of varying ratios of flour to water on the diastatic 

activity of a wheat flour. 

Weighed Calculated 

as CuoO Weight of 

CujO per 
I. ' Gram Flour 
Grams 

.2680 
.2900 
.3430 
.3348 



.3340 



Sample 
Number 


Weight of 
Flour 
Grams 


H.O 

Volumes 
cc 


Ratio 


per 2.5 
Grams Flo 
Grams 


1. 


50. 


27.5 


1 :0.55 


.0675 
.0665 


2. 


50. 


35.0 


1:0.70 


.0727 
.0723 


3. 


50. 


50.0 


1:1.0 


.0862 
.0853 


4. 


10. 


25.0 


1:2.5 


.0835 
.0835 
.0827 


5. 


10. 


75.0 


1 :7.5 


.0838 
.0831 
.0836 


6. 


10. 


100.0 


1:10,0 


.0833 
.0836 
.0830 
.0839 


7. 


10. 


400.0 


1 :40.0 


.0835 
.0835 
.0820 



.3338 



.3340 



The low results for samples numbers 1 and 2, table XIII. are prob- 
ably due to an insufificiency of water to allow complete enzymatic ac- 
tivity. The normal dough for this flour would require 29.5 cc of water 
for 50 grams of flour, which is less than the amount added to sample 
number two. It must be understood, therefore, that diastatic action 
is not complete in a normal dough under average bake shop conditions. 
The result for diastatic activity in sample number three on the other 
hand, is too high by about 3. percent, yet it falls within the range of 
values as determined by the other samples. Considering, however, 

^ 46 



the necessary inaccuracies of method in controlling temperatures, in 
doughing up the flour samples, in weighing and dividing the dough, 
and especially in the time required to disperse the dough and dissolve 
out the sugars before the enzymic activity can be inhibited by clari- 
fication, the agreement with the samples numbers 4 to 7 is about as 
close as could be expected. Attention might be called to the fact that 
the results as expressed in column five contain the errors of the actual 
determinations multiplied four times. 

The excellent agreement of the results for autolytic digestion of 
the flour with water in ratios of 1 :1 or more, over so wide a range of 
concentrations, eliminates one of the most troublesome factors in a 
determination of this nature. It enables one to add other reagents or 
to vary the total valume of the solution at will and yet have only the 
effect of the one desired variable imposed on the results. 

It is evident here that the enzyme and its substrate exhibit an in- 
timate relationship. The large and practically constant excess of 
starch substrate is available for the relatively small amount of en- 
zyme which exists within the plant material constituting the flour 
particles and which is held in close contact with the starch granules 
on which it must act. When sufl^icient water has been added to prop- 
erly hydrate the bio-colloids and enable the enzymes to function, any 
further increase in the amount of water has but little effect, the 
enzymes having been adsorbed to the substrate and not immediately 
subject to dilution. This is not the case with the solutions of "puri- 
fied" and dissolved diastatic preparations or extracts, which do show 
some concentration effect because of their suspension throughout the 
solution. The results of Thatcher and Koch described above, as well 
as the work of nearly all of the other investigators of diastase, have 
shown that a part, never all, of the diastatic activity could be extracted 
from flour as a solution. But these extracts do not show normal, i. e. 
natural, diastasis as in the plant tissue. Frequent shaking and pro- 
longed digestion are necessary for such a removal of enzymes. It 
should be stated here in this connection that too frequent and violent 
shaking of the flour-water suspensions used in this work decreased 
considerably the quantities of maltose produced per unit of time, and 
the supernatant liquid evidenced increasing diastatic power. It was 
for that reason that the suspensions vvere agitated only at fifteen 
minute intervals by rotating the containing flasks. 

But by far the more important contribution of this set of results to 
the problem in hand rests on the demonstration that autolysis of flour- 
water suspensions in ratios around 1 :10 as carried out in this series of 
experiments can be considered as furnishing a logical laboratory basis 
for the measurement of diastatic capacity of the flour when mixed in a 

47 



dough. The temperature is taken as the average accepted fermenting 
temperature for ordinary bake shop practice. The time may be con- 
trolled at will. The pH of the digestion mixture is controlled by the 
Hour, usually between 5.7 and 6.1, and is close to that at which the 
dough fermentation normally begins. The acidity which develops as 
fermentation proceeds in the dough gradually increases to a pH value 
between 4.8 and 5.4, at w"hich point the diastatic activity is at or very 
near its maximum. Sherman and Thomas (1915), and Sherman, 
Thomas, and Baldwin (1919). showed that the protective action and 
stabilizing effect of buffer salts on diastase is very marked both with 
respect to concentration and temperature. This buffer effect undoubt- 
edly accounts in part for the stability of the enzyme rate by the meth- 
od of measurement employed here. This is also shown further on by 
the data on relation of pH to diastatic activity. 

The relations between avitolytic measurements and diastatsis in the 
dough during a normal fermentation are discussed further in connec- 
tion with measurements of maltose production in doughs. 

It would seem advantageous then from the standpoint of sugar 
production to mix doughs with the maximum amount of water which 
they will carry and still work well. The machinery designed for the 
modern large baking plants will handle doughs with absorption as 
high as 70%, which allows practically maximum maltose production 
by diastatic action. 

Method for Measuring Diastatic Power of Flour. As a result of the 
data obtained up to this point a definite method was developed for the 
determination of diastatic power in flour. This procedure was used 
with variations in time, temperature, or pH, as the case might require, 
for all the experiments which follow. 10. gram samples of flour are 
weighed out and transferred to 250 or 300 cc Erlenmeyer flasks. These 
are placed in a water thermostat and brought to a temperature of ex- 
actly 27° C. A flask containing a suft'icient volume of distilled water 
is also placed in the bath and kept at 27° C for subsequent use. By 
means of a pipette 100. cc of the distilled water are run into the sam- 
ple, while rapidly rotating the flask to obtain a thorough suspension 
of the flour. The last few cc's in the pipette are allowed to rinse the 
material down from the sides of the flask. The flask is quickly re- 
placed in the thermostat, stoppered loosely, and allowed to remain 
exactly 60 minutes. A few" minutes after starting the digestion the 
flask is rotated to stir up the suspension and hasten the equalization 
of temperatures, and the shaking is repeated at fifteen minute inter- 
vals. At the end of the digestion period the contents of the flask 
are quickly rinsed into a 200 cc volumetric flask, diluted to about 175. 
cc and clarified. 

48 



To clarify, first make sure that the solution is neutral or slightly 
alkaline. Five drops of 0.04% Thymol Blue serves as a convenient 
indicator, appearing cream-yellow in color when neutral, or blue when 
slightly alkaline. Add 3 cc of a 15% solution of sodium tungstate 
(Na2W04.2H20)and mix thoroughly with the flour suspension. Then 
add, drop by drop, from a 1 milliliter graduated pipette, with con- 
stant shaking, sufficient concentrated HoSO^ to turn the indicator a 
decided pink color, with two or three drops in excess. Four-tenths 
of a milliliter (0.4 cc) are usually sufficient if the original flour suspen- 
sion was nearly neutral. This clarification likewise serves to stop 
the enzymatic activity and prevents further change in sugar content. 
Dilute to the mark, shake thoroughly, pour into centrifuge cups, and 
whirl for about five minutes. By means of a calibrated pipette trans- 
fer 50. cc of the clear supernatant liquid to a 400. cc pyrex beaker 
for the determination of reducing sugars by the Munson-Walker offi- 
cial method. 

A blank is also run at the same time as the sample to correct for the 
natural reducing sugars in the flour. It likewise gives a measure of 
these reducing sugars. To prepare the blank, mix 100. cc of water at 
27°C and 10. grams of flour and immediately inhibit diastatic activity 
by clarifying with the sodium tungstate in the manner just described. 
The blank determination is then carried out in the same manner as the 
samples except that the addition of 0.4 cc of concentrated HgSO^ is 
omitted on dilution to volume. It does no harm to allow the clari- 
fied solution in the beakers to stand an hour or two until other samples 
are ready. When making the Fehling reduction the excess acidity 
of the solution can conveniently be neutralized by using a predeter- 
mined number of drops of strong NaOH. Following the reduction 
of the Fehling solution the CugO is filtered, washed, dried, and 
weighed in the gooch crucibles. Subtract the weight of the CuoO 
corresponding to the blank from that of the sample. The result is the 
CuoO corresponding to the maltose produced by the diastase in 2.5 
grams of flour. This value for anhydrous maltose as found from the 
tables, multiplied by 4. gives the diastatic power per 10. grams of 
flour. This procedure has been found by many repeated trials on dif- 
ferent flours to give accurate results which could be duplicated with- 
out difficulty. 

The temperature of 27° C is chosen because that is very near the 
average of the temperatures at which doughs are fermented in com- 
mercial practice. The proofing temperature, usually around 
32.5°C is varied widely in practice, and continues at that temperature 
but a short time compared to the total fermentation time. Ten grams 
arc chosen for a sample because the reducing sugars produced give a 

49 



convenient weight of CuoO on the asbestos mat. The 200 cc final 
volume is selected because it conveniently fills two 100. cc centrifuge 
tubes which balance each other. However, no error is introduced by 
a change of final volume to a 250, 300 cc or 500 cc flask, and the latter 
is sometimes desirable when working with a sample of unusually high 
diastatic power, such as a malt flour. 

The application of this procedure to a determination of malt prep- 
arations for use in panary fermentation require some special consider- 
ations, and will be left for discussion further on in this paper. 

Effect of Time and Temperature on Activity of Flour Diastase. 4. 
Temperature. The factors of greatest importance in the limitation 
of enzymatic activity have been shown by many investigators to 
be those of temperature, hydrogen or hydroxyl-ion concentration, 
and of time. Of these, the temperature factor will be considered first 
because it is by far the most significant in determining the produc- 
tion of maltose by the action of diastase under the conditions obtain- 
ing in a bread dough. Because of the very high rate of autolytic dias- 
tasis in these flour-water suspensions at temperatures near the range 
of optimum temperatures it was difficult to prevent large errors in 
the results between 50.° and 70.°C. Slight variations in temperature 
for the first few minutes of digestion vitiated the value of the deter- 
mination. Preliminary experiments in connection with temperature 
control had shown that a few minutes heating of the dry flour in flasks 
in the water bath had no effect on the diastatic activity providing the 
temperature was not above 65°C. Prolonged heating, however, pro- 
duces a very slow reaction due no doubt to the partial vaporization of 
the moisture in the flour and its condensation on the sides of the flask 
with consequent wetting of some of the flour. After an hour of heat- 
ing at 70°C a sample of flour was found to increase slightly in reduc- 
ing sugars and to decrease in diastatic power. 

The procedure followed in this series was to first bring the water 
thermostat up to the temperature desired. The 10. gram samples of 
flour, in loosely stoppered erienmeyer flasks, were placed in this bath 
about ten minutes before the water was added. Exactly 100. cc of 
distilled water in 150 cc. erienmeyer flasks, were heated to the desired 
temperature in the water bath and poured quickly on to the flour 
sample. A sensitve 100° C thermometer was placed in the flask before 
adding the water. The temperatures resulting from the mixing of 
flour and water were usually a few tenths of a degree below that de- 
sired. They were quickly corrected by rapidly rotating the flask over 
the hot spot of a wire gauze heated by a bunsen flame, and the flask 
then replaced in the water bath. At the end of the desired time the 
flask was removed from' the bath, transferred to a 200 cc volumetric 

50 



flask, clarified, cooled when necessary, diluted to volume, centrifuged, 
and the reducing sugars determined as described above. Figure 1 
shows graphically the nearly autocatalytic nature of the temperature 
curve for one hour diges'tion. Three hour digestions gave a curve of 
the same kind. The values from which the curves are plotted are ex- 
pressed in Table XIV as grams of maltose produced by the autolytic 
diastasis in 10. grams of flour. These values are obtained from the 
weights of CujO corresponding to the 50. cc alicjuots of the digestion 
solution. The blank determination, representing the natural reducing 
value of the extracted flour-sugars, in terms of Cu^O, was subtrated 
from the total weight of CU2O as obtained from the auto-diastasis. 
The difference gave the cuprous oxide equivalent to the maltose pro- 
duced by diastasis alone. The corresponding weight of maltose found 
from the Munson-Walker table was multiplied by the necessary 
aliquot number to obtain the value per ten grams (total) of sample. 
Each value is the average of at least three reducing sugar determina- 
tions for that time and temperature. The maltose values are calcu- 
lated as "diastatic" maltose, in grams. 



TABLE XIV. 

The relation of temperature to the activity of wheat flour diastase. 

10. grams flour No. 1009 used for each sample. 

Weight of Maltose 
from Diastasis of 
Temperature 10. grams Flour. 

Degrees 1 Hour Digestion 

Centigrade Grams 

0. .0386 

27. .2118 

25. .3238 

55. 1.1378 

60. 2.1396 

63.5 2.9067 

65. 1.6408 

67. .7698 

70. .4016 

75-76 .0922 

*82-83 .0244 



*Gelatinized. 



The maximum production of maltose was 29% at 63.5°C. But this 
temperature ( 146.3° F) is probably never reached in a dough during 
a normal fermentation, and only for a few minutes while baking. 



51 




Temprmture 



FIGURE 1. 
The effect of temperature on the activity of wheat flour diastase. 

5. Time. The relationships between activity, temperature, and 
time, can be conveniently considered together. Since the rate of dias- 
tasis is so enormously increased at higher temperatures, the curves for 
the production of maltose with time must show a corresponding in- 
creased initial rate at higher temperatures. Consequently the time 
curves for several temperatures were determined and are plotted in 
Figure 2. The data corresponding to these curves is tabulated in 
Table XV in groups of results, one set for each curve. To simplify 
tabulation the total* values per 10. gram sample are calculated from 
the actual weighings as described for the values in Table XI\\ The 
last set of data in Table XV is obtained on a different flour (our 
laboratory sample No. 1) and is also included in Figure 2 to show 
the similarity of the results for a flour of lower diastatic power. 

TABLE XV. 

The variation of diastatic activity in wheat flour with time. 

Grams of Maltose Produced by Diastase in 10. grams of Flour 
Flour Time in Minutes 



Number 


Temp. 


30 


60 


120 


180 


240 


300 


360 


1009 


O.'C. 




.0383 




.0702 








1009 


27.x. 




.2114 


.2942 


.3578 


.3950 


.4420 


.4650 


1009 


35.°C. 


.2334 


.3235 


.4497 


.5181 


.5832 




.6230 


1009 


55.*C. 


.5608 


1.0380 


1.2464 


1.3862 


1.4916 


1,4820 




1009 


64-65. °C. 




1.5402 


3.0796 


2.6718 








1009 


69-70.°C. 




.3801 


.8225 










1 


27.°C. 


.0725 


.0963 


.1313 


.i423 


.1632 


,1741 


.1870 



52 




60 /ZO 180 

Time - minutes. 



FIGURE 2. 

The change of activity of wheat fiour diastase with time, 
at different temperatures. 



The curves drawn from the data in Tal)lc X\^ rescmltle very closely 
those which Collatz and Bailey obtained for the increase of conductiv- 
ity in flour extracts due to the action of the enzyme phytase on the 
l)h\'tin. It w^ould appear probable that these two enzymes exhibit a 
parallel activity for a given flour at any particular temperature. 

The rate of maltose production has reached practically a constant 
value, for ordinary fermentation temperatures, between two and three 
hours diastasis. In the case of Flour No. 1, Table XV\ the graph for 
maltose production with time is nearly a straight line from two to 
twenty-four hours. 

53 



6. Acidity. The diastases of many biologically different materials 
of both plant and animal origin, have been investigated and reported 
in the literature. The temperature optima vary considerably for dif- 
ferent samples, no doubt due to adaptations to environment. The 
agreement, however, of the optima for hydrogen ion concentrations 
of most of the diastases reported seems to point to a general relation- 
ship of activity. Because of the close relationship between the barley 
and wheat grains the diastase of wheat would be expected to show an 
optimum activity at a pH of 4.7 to 5.0, corresponding to that for the 
diastase of malted barley reported by Sherman and Walker, and Sher- 
man, Thomas and Baldwin. 



TABLE XVI. 

The influence of hydrogen ion concentration on the activity of 
wheat flour diastase. 

10. gram samples. Flour No. 1009. 1. hour digestion @ 27 C. 













Corre- 








Weight of 


sponding 








CU2O in 


Milligrams 


Weight of 
Maltose in 






pH After 


Weight 


Weight 


Milligrams 


Acid ( 


or Alkali 


1 Hour 


per 50. cc per 10 g. 


per 10 g. 


Added 


Digestion 


Aliquot 


Flour 


Flour 


25. cc 


N/10 HCl 


1.946 


15.7 


62.8 


+. 


10. cc 


N/10 HCl 


2.775 


27.5 


110.0 


34.8 


15. cc 


N/25 HCl 


3.522 


78.4 


313.6 


196.1 


10. cc 


N/25 HCl 


4.006 


109.6 


438.4 


295.1 


10. cc 


N/25 HCl 


4.034 


111.8 


447.2 


302.2 


7.5 cc 


N/25 HCl 


4.399 


115.4 


361.6 


313.5 


5.0 cc 


N/25 MCI 


4.808 


116.5 


465.9 


316.8 


3.0 cc 


N/25 HCl 


5.112 


111.3 


445.2 


300.6 


2.5 cc 


N/25 HCl 


5.195 


112.6 


450.4^ 


304.7 


0.0 




5.691 


83.7 


334.81 
335.4/ 


213.4 


0.0 




5.742 


83.8 


2.5 cc 


N/25 NaOH 


6.352 


45.1 


180.4 


90.7 


5.0 cc 


N/25 NaOH 


7.023 


34.7 


138.8 


57.4 


7.5 cc 


N/25 NaOH 


7.527 


25.2 


100.8 


27.4 


10.0 cc 


N/25 NaOH 


9.067 


19.5 


78.0 


9.9+. 1 


5.0 cc 


N/25 NaOH 


9.134 


16.5 


66.0 


-I-. ' 


6.0 cc 


N/10 NaOH 


9.968 


16.0 


64.0 


+. 


Blank 






14.1 


56.4 





Figure 3 is a graphical representation of the data in Table XVT 
and shows the production of maltose by diastatic enzymes with vary- 
ing concentrations of hydrogen and hydroxyl-ions, in terms of pH. 
To obtain the data from which Table XVI is compiled, pairs of sam- 
ples were run simultaneously. The regular procedure described 

54 



above was used for each sample in which the reducing sugars were to 
be determined, acid or alkali being added as shown in the table at the 
beginning of the digestion to produce the desired pH. The other, or 
check sample was treated in exactly the same manner up to the end 
of the digestion period, when instead of clarifying, the suspension was 
thoroughly shaken up, poured directly into centrifuge cups, centri- 
fuged, and the supernatant liquid at once subjected to pH measure- 
ments. In this manner the resulting pH at the end of the one hour 
digestion was measured for each sample. For convenience in drawing 
the curves in Figure 3, the weights of CugO equivalent to the diastatic 
activity were used to prepare the graph (Figure 3), rather than the 
corresponding weights of maltose. This was because the lower mal- 
tose values were too small to be accurately determined from the mal- 
tose tables. However, the results are expressed in milligrams of mal- 
tose actually produced by the diastase in ten grams of flour. The cal- 
culation of results in this manner have been described above. 




The relationship between the activity of wheat flour diastase 
and the pH of the medium. 



The diastatic activity of wheat exhibits practically the same maxi- 
mum pH as that found for barley malt. This maximum occurs at a 
pH of 4.7 to 4.8, with a broader range of maxima between 4.0 and 5.3. 

55 



The rapid decrease in the production of maltose in the ranges of pH 
from 5. to 6.5 may be of particular significance in the fermentation 
of different grades of flour. It may also account for some of the dif- 
ferences in baking strength which become apparent with aging of the 
flour. 

The hydrogen-ion concentration of the normal bread dough when 
mixed is very approximately that of the flour, usually slightly higher, 
and in the case of the flours here used varies from a pH of 6.15 to pH 
5.6. The optimum pH for diastatic activity is never reached in the 
short fermentation, straight dough process, for a normal dough, and 
, not often in sponge doughs. Even in the latter case such a high acid- 
ity is obtained only after six or more hours of fermentation, at which 
time the diastase has suffered greatly in loss of activity due to changes 
of hydration of the colloidal proteins. As the fermentation advan- 
ces the hydrogen-ion concentration slowly increases, until an appar- 
ent miximum is reached somewhere around a pH 5.4 to 5.2, when the 
dough is ready for the oven. This point may well be that condition 
in which the COg produced by the zymase of the yeast cells has sat- 
urated the dough. The eft'ect of this increase of acidity may then be 
regarded as reciprocal, first the increasing hydrogen-ion concentration 
from a pH of 6. to pH 5. has the effect of nearly doubling the maltose 
produced per unit of time. The maltose in turn becomes immedi- 
ately available as food ft^r the yeast cells, which thereby are enabled to 
renew their fermentation activity and increase the rate at which the 
CO2 is produced for the aeration of the dough. This increasing activ- 
ity is further augmented by a temperature increased during proofing 
of the panned loaves ; this furnishes part of the explanation for the 
rapid rise of the loaf in the pan just before baking. The yeast activity 
is likewise stimulated by the same increase in temperature and acid- 
ity. That it is the function of the diastatic enzymes present, and not 
of the sugar originally added to the dough, which largely controls the 
proofing action, is shown further on in connection with experiments 
on the diastatic action in actual doughs. 

We should not lose sight of another important factor in the latter 
stages of fermentation and proofing of the dough. That is the soft- 
ening effect of the proteoclastic agents on the gluten. The gluten 
suffers more or less rapid proteolysis : becomes softer and less elastic. 
Thus the increasing rate of carbon dioxide production is able to 
"raise" the dough easier and more cjuickly. 

Buffer Action. The buffer effect of different flours must likewise 
be taken into consideration in this connection. The highly refined 
flours, such as patents, generally show a lower buft'er value than the 
less highly refined, or clear flours. Therefore, the maximum acidity 

56 



should be reached more quickly during the fermentation of a patent 
flour, resulting in a more rapidly increasing rate of fermentation and 
consequently a 'better aeration. On the other hand the better quality 
of gluten in these grades of flour requires more time for its proper 
"ripening," is more tenacious and elastic, and so requires a greater fer- 
mentation activity to overcome its tightness. Also, its greater gas 
retaining capacity helps to make up a combination of strength char- 
acteristics which taken altogether, form the desirable qualities for the 
production of a good loaf of bread. 

The flours of poorer grade, such as high percentage straights and 
the clears, though made from the same w^heat, usually show a higher 
buffer value. The natural efifect of their bufifer salts is therefore to re- 
quire a greater amount of acid formed by fermentation before the 
proper pH of the resulting dough is reached for maximum diastatic 
activity. It may be that the same concentration of hydrogen-ions, 
namely 10"^' is not reached in the doughs from low grade flours. The 
decreasing quality of the glutens, however, in the lower grades of 
flours, is oftener the limiting factor because of their very poor gas re- 
taining capacity, and the}^ are able to show but little improvement by 
the addition of acids and diastatic enzymes. Thvis before the inter- 
relationships of these three factors, gluten strength, proteoclastic 
activity, and diastatic activity can be more accurately explained, it 
becomes necessary for further study on each one of them. 

The above consideration of buffer values of flour and hydrogen-ion 
concentrations in the dough demonstrate the desirability of deter- 
mining these values for each of the flour samples used for this investi- 
gation. The results of such a set of determinations are given in part 
here, and afford a confirmation of those of Bailey and Peterson (1921). 
The buffer values of the fourteen flours were determined by making 
water suspensions of each sample, of 1 : 5 concentrations, as recom- 
mended by Bailey and Peterson. These were kept at 25. °C and 
shaken at intervals for one hour. The suspensions were then centri- 
fuged without filtering, and to 25. cc aliquots of the centrifugate were 
added varying amounts of .02 Normal HCl or NaOH. The resulting 
pH was determined at once in Bailey electrodes by potentiometer 
measurements. For the sake of brevity only one of the fourteen sets 
of readings is included in Table XVII. Figure 4 includes the graphs 
of three sets of values of widely different grade. The addition of .02N 
acid was carried up to 40. cc to better show the differences in the 
shapes of the curves. 



57 



TABLE XVII. 



The Buffer Value of Flour Sample No. 1001. 



Acid 


or Alkali 


Added to 25 




of 1.5 Flour Extract 




12.5 cc. 


.02N HCl 




10.0 cc 


.02N HCl 




7.5 cc 


.02N HCl 




5.0 cc 


.02N HCl 




2.5 cc 


.02N HCl 




0.0 cc 






2.5 cc 


.02N NaOH 




5.0 cc 


.02N NaOH 




7.5 cc 


.02N NaOH 




10.0 cc 


.02N NaOH 




12.5 cc 


.02N NaOH 



pH. 

2.519 
2.654 
2.925 
3.388 
4.150 
5.816 
7.371 
9.045 
9.775 
10.253 
10.617 




cc %o *«« 



Figure 4. 

The buffer curves of three flour of widely different baking value, 

acid range. 

58 



Because of the large number of buffer values falling within rather 
narrow limits, and the crossing of the curves on the alkaline side, the 
complete curves cannot all be drawn in. 

Figure V afifords a comparative idea of the buffer values in the acid 
range, for each of the flours used. The height of the columns repre- 
sent the relative change in pH resulting from the addition of 20. cc of 
.02N HCl to 100 cc of the centrifuged 1 : 5 flour-water extract. The 
arrangement of flours is in the order of their baking value. For con- 
venience the series of fourteen samples have been arbitrarily divided 
into three classes. The columns in black represent the group of 
flours which would ordinarily be considered by the baker as those 
possessing good baking qualities. The crossed lines indicate those 
flours pf rather poor quality, but which can nevertheless be used for 
bread, and especially so when blended with stronger flours. The col- 
umns in diagonal lines represent those flours which are of such poor 
quality and low baking value that they could not be used for market- 
able bread in this country. While flour No. 1013 might be included 
in the second group because of its relative strength, its poor color 
would make it of doubtful value for blending. 




_ 00 f>4 o< 
I a § ^ 



*<) "O <a 
^ c:^ ^ 



Hard Wheat Patents 
and Straight Flours 



Soft 

Wheat 

Flours 



c^ e>» St- >• 

Clear Grade 
Flours 



Figure 5 

The relative change in pH produced by addition of 20 cc HCl to 
100 cc 1 :5 flour-water suspensions. Flours arranged in the order of 
their baking value. 



59 



There is no apparent relati(.)nship shown by the values for buffer 
action and the corresponding diastatic activity of the flour as given in 
Table XVTII. This could only be shown by a set of determinations of 
both pH and diastatic activity in the actual dough during fermenta- 
tion. The changes of pH with progressive fermentation of the doughs 
made from this set of flours are given in another report. Work is 
now in progress in several quarters on the development of a special 
form of electrode for the determinaticm of hydrogen-ion concentration 
in doughs. Experiments in the laboratt)ries of one of the larger 
American bakeries have demonstrated the commercial application of 
such electrometric measurements for the control of dough fermenta- 
tions. 

Relative Diastatic Powers of Flour Samples. The results on deter- 
minations of the diastatic powers of each of the fourteen flours are 
given in Table XVIII. 

The regular procedure described herein was used, namely, one hour's 
autolysis of a 1 :10 flour-water suspension (a) 27° centrigrade. 

TABLE XVIII. 

Comparative measurements of diastatic power on fourteen samples 

of flour. 

Diastatic Power 
Maltose by- 
Diastase in 
10. g. Flour 
Milligrams 
248.2 
186.5 
34.8 
145.0 
131.9 
105.7 
123.6 
304.1 
211.8 
92.6 
51.7 
132.9 
132.6 
116.2 

The relation of the respective diastatic powers of these flours to 
their baking strength can be more easily shown in Figure 6, where the 
arrangement of flours is according to baking strength, the height of 
column representing the milligrams of maltose produced in one hour 
by 10. grams of flour. Each value is the average of three or more 
determinations for each flour. With the exception of the very low 
grade flours these samples show diastatic powers which are a fairly 

60 





Active Sample 


Inactive Sample 


Flour 


Weig-hed Cu.O 


Weighed Cu-O per 


Sample 


per 2.5 g. Flour 


2.5 g. Flour 


No. 


Grams 


Grams 


1001 


.1031 


.0223 


1002 


.0762 


.0148 


1003 


.0226 


.0091 


10(W 


.0638 


.0155 


1005 


.0626 


.0185 


1006 


.0513 


.0154 


1007 


.0563 


.0145 


1008 


.1146 


.0162 


1009 


.0833 


.0140 


1010 


.0474 


.0157 


1011 


.0254 


.0065 


1012 


.0529 


.0085 


1013 


.0523 


.0080 


1014 


.0553 


.0161 



good index of their general fermentation characteristics. That is, the 
behavior of the dough in the latter stages of fermentation, and the 
"spring" in the oven, show that the diastatic power of the flour as 
measured by the method here employed does furnish an indication of 
the general strength characteristics of that flour, especially with regard 
to volume and texture. The quality of the gluten, however, must be 
sufficiently high to conserve the value of the diastatic action and make 
a good loaf possible. It would be necessary to study many more 
samples of flour before extending such an observation to the status 
of a general conclusion. 

From these samples of flour, difTering so widely in type and grade, 
no conclusions can be drawn as to the relationship betw^een diastatic 
activity of a flour and climatic factors in the growth of the wheat. 
Only flours produced by a uniform milling practice from wheats typi- 
cal of dift'erent growing regions could be used to furnish information 
of such a nature. 



dOO — 



200 




•s. 00 <V «( )9 
O 5 "5 v S 
^ 5 ^ ^ 5 



Hard Wheat Patents 
and Straight Flours 



^ ^ ^ 

5 ^ <5 

Soft^ 
Wheat 
Flours 



R ^ I ^ 
? ^ ^ ^ 

Clear Grade 
Flours 



Figure 6 
Relative diastatic powers of fourteen flours, in order of their 

baking value. 
61 



. Diastatic Activity During Fermentation of the Dough. The opin- 
ions of several of the earlier investigators concerning the significance 
of diastatic enzymes in a dough during the latter stages of fermenta- 
tion have been reviewed in the historical part of this paper. 

The early English practice, revived during the late war, of ferment- 
ing doughs without added sugars would throw the burden of sugar 
production entirely upon the diastase of the flour unless other dias- 
tatic material, such as malt, were added. The production of carbon 
dioxide by the zymase of the yeast cells must continue with some 
regularity throughout the frementation period in order that the dt)ugh 
mass may be properly aerated. 

The increased enzymatic activity during the forty-five to sixty 
minutes in which the dough is allowed to rise in the pan before bak- 
ing is of especial importance. It is th^s final period of aeration which 
determines to a large extent the texture and "lightness" of the loaf. 
The gluten, however, must iiave the requisite quality. The proofing 
period is usually carried out at a temperature of eight to ten degrees 
higher than that of the fermentation up to this j^oint. It is <»bser\ed 
from Figures 1 and 2 that this increase in temperature from 27° to 
35°C increases the diastatic activity nearly 30 percent. In actual 
baking practice, however, the rise of temperature in the interior of 
the proofing loaf is very slow and a temperature of 33 to 35°C is prob- 
ably not reached until near the end of the ])roofing period. 

If the yeast cell is to increase its zymase activity during })roofing 
in order to provide the carbon dioxide necessary to properly raise the 
dough and shape the loaf, there obviously must be a sufficient supply 
of available sugar. If the present commercial practice of straight 
dough fermentation there is rarely a sufficient supply of sugar added 
at the time of mixing to carry the yeast activity throughout the fer- 
mentation. The diastase of the flour, if present in sufficient quan- 
tity, must carry a part of the load. The addition of a fresh supply 
of sugar by mixing in with the dough when ready for proofing is not 
practical for several reasons, and a too large supply at the tiiuc of 
mixing the dough often stimulates the yeast activity far beyond that 
desired. The yeast must then depend largely upon the sugars produced 
by the diastase for its fermentation activity during the proofing. This 
sugar, in the form of maltose, is readily split down to dextrose bv the 
maltose of the yeast, and so is immediately available as substrate for 
the yeast zymase. 

We might also expect that there would be a slight difference shown 
m the rate of carbon dioxide production between the action of yeast 
fed on sucrose added at mixing, and on maltose produced by diastase 
in the dough. The latter may be regarded as a steady rate of maltose 

62 



formation within the colloidal medium immediately surrounding the 
yeast cells. The distance the maltose must diffuse to reach the ac- 
tive yeast surface is thus at a minimum. On the other hand the util- 
ization of sugars added to the dough must decrease in rate as the dif- 
fusion distance from the active yeast interface increases. This would 
further lead to the opinion that in theory the substitution of the req- 
uisite diastatic enzymes for a part of the sugar usually added to the 
dough at mixing should produce a better and more uinform fermenta- 
tion, and in general a better loaf. Some sugar of course should be 
present at the beginning of the fermentation to stimulate the yeast 
activity and supply food for the early fermentation, unless the whole 
fermentation period be lengthened accordingly. After that time the 
diastase should have reached nearly a constant rate of maltose produc- 
tion and should, therefore, be able to supply the necessary sugar in a 
more available form. 

In practice, however, the addition of considerable amounts of dias- 
tatic malt preparations, e. g., malt flours or malt extracts, is compli- 
cated by the excessive activity of the proteoclastic enzymes which 
they usually contain. These malt preparations likewise often contain 
pigmented material which is not destroyed during fermentation. In 
fact some of the pigmented particles appear to be so aft"ected by fer- 
mentation that they are made soluble and tend to diffuse into the 
dough, darkening the color of the linished product. Here again the 
importance of the proteoclastic enzymes and the necessity for their 
measurement and control, often overshadow the practical application 
of diastatic malt products in the bakery. 

It appeared highly desirable, therefore, to make some measurements 
of sugar content and diastatic activity in doughs during the normal 
fermentation. Regular test doughs were accordingly mixed, omitting 
either the yeast, or sugar, or both, depending upon which type of re- 
sultant sugar content was to be measured. The lengthy method of 
extracting the sugars from these doughs with alcohol, followed by 
evaporation, dilution, clarification, etc., is hardly suited to the rapid 
determination of total and reducing sugars in several doughs at short 
intervals. The procedure which had been previously used for the 
measurement of diastatic activity in doughs suggested itself as a possi- 
ble basis for a satisfactory method. Several trials at 30 minute inter- 
vals during the fermentation of a dough without yeast, gave results 
which when plotted in terms of sugars produced per unit of time, 
showed a smooth curve corresponding roughly to the enzvme curves 
of Figure 2. The curves for a number of flours indicate that the 
method will at least give comparative results for each type of sugar 
content and for each flour. 

63 



The procedure finally used for these determinations was as follows: 
A two-loaf dough was mixed as for a baking test in the manner pre- 
viously described. This was taken from the mixer and divided into 
two equal batches, one of which was fermented and baked, and used 
as a control dough, the other being handled in the same manner ex- 
cept that from it samples were taken at desired intervals. From 
these samples two ten gram portions were weighed off, one taken im- 
mediately for sugar analysis, and the other used for the determination 
of total solids. The sample to be analyzed for sugars was at once 
shaken up in a 500 cc bottle, with about 100 cc of distilled water to 
which two or three drops of 50% NaOH had been added, until the 
gluten had been dispersed and the sugars dissolved out. This dough 
suspension was poured into a 300 cc volumetric flask, clarified and 
treated as described above in the regular procedure. In some cases a 
50. cc aliquot of the clear centrifuge was taken for hydrolysis and 
determination of total sugars by the A. O. A. C. method. The results 
for the total sugars while substantiating the conclusions to be drawn 
from those of the reducing sugars, do not add materially to the infor- 
mation desired, and are not included here, as they were not obtained 
in complete sets for all samples. 

Many determinations were made in this laboratory on the effects of 
diastatic action during the fermentation of doughs. Different kinds 
of diastatic malt preparations, in varying amounts were used in the 
doughs, with and without other sugars, and with and without yeast. 
The results are included in a separate report on flour strength as in- 
fluenced by the addition of diastatic ferments. (Collatz, 1922). Only a 
few examples have been selected from this available data to show the 
role which diastase plays in panary fermentation. These are designated 
by Roman numerals from I to VIII, corresponding to the curves in 
Figures 7-10. The experiments with these doughs were made in pairs, 
one dough of each pair was made up with the usual amount of yeast, 
the other without yeast. In this manner the relation of sugar pro- 
duced by the diastase to that required by the yeast fermentation 
under the same conditions to temperature is clearly shown. Dough I 
was the regular baking test dough, containing 3.0% of sucrose and 
2.5% yeast. The resulting loaf was above the average, though slightly 
overproofed. The texture was fair, the volume large (2110. cc), and 
the color good, but the grain was a bit too open because of overproof. 
Dough II was the same as No. I except that the yeast had been 
omitted. Dough III was mixed without sugar, but otherwise the 
same formula as Dough I. The fermentation time was necessarily 
longer. The resulting loaf was of very good quality, of very fine 
grain, good texture and an even but strong break. The color of crust 

64 



was fair, but considerably lighter than that of loaf I, as would be 
expected from the lack of sugar. The volume likewise was less, be- 
ing only 1870. cc. Dough IV was the same as No. Ill, omitting the 
yeast. The supply of flour No. 1009 was exhausted at this point and 
another sample was obtained from the same mill. This second 
sample, designated as No. 1009A was somewhat inferior in strength 
ajid color, but the behavior of its diastatic enzymes were practically 
the same as those of No. 1009. Diastatic activity and reducing sugar 
determinations for doughs mixed with and without sucrose showed 
practically the same curves as those for doughs Nos. 1 to IV. Flour 
No. l609A was, therefore, used for doughs V to VIII. Dough V 
was mixed with 2.% of a malt flour as a source of added diastatic en- 
zymes, but no other sugar was added. Although the amount of this 
particular malt flour was somewhat in excess, as indicated by the 
grayish color of the crumb and the slight coarsening of the grain, the 
effect of the added enzyme is clearly shown in curve V, The volume 
of the loaf was increased 12% over that of a standard dough contain- 
ing sucrose but no added malt diastase. The further discussion of 
this phase of the question of added diastatic enzymes is considered in 
detail in the second report. (CoUatz, 1922). Dough VI was mixed with 
the 2.% of malt flour but contained no yeast. Dough VII : The total 
amount of maltose produced in dough V by diastatic action of com- 
bined wheat flour and malt flour was calculated back into terms of 
anhydrous maltose per unit weight of flour. This percentage of an- 
hydrous c.p. maltose was then added to Dough VII, without other 
sugars or diastatic products. The resulting loaf bore the same rela- 
tion to the standard loaf as in the case of Doughs Nos. I and II, 
namely, a finer grain, good texture, lighter color of crust, and slightly 
smaller volume. Table XIX is furnished here as an example of the 
data obtained for each dough, and comprises the basis from which 
the points were plotted in Curve III, Figure 8. To save space and 
to avoid multiplicity of headings the data for the complete set of 
curves, Figures 8, 9, 10 and 11, are abbreviated and grouped in Table 
XX. 



65 



TABLE XIX. 

Reducng Sugars and Diastatic Activity During the Fermentation of 
a Dough. Mixed Without Sugar. Curve III. 

Weight of Reducing 

Total Weight of Sugars as 

Solids CU2O per Maltose 

Sampled Total Temper- Weight of per 10. g. 10. g. per 10. g^ 

At Time ature Dough Dough Dough Dough 

Minutes Centigrade Grams Grams Milligrams Milligrams 

Mixing 0.0 27.° 530.0 5.5720 207.0 156.0 

1. hour 60.0 27.5° 274.4 209.5 

1st Punch 177.0 27.5° 526.0 5.5222 269.4 205.5 

2d Punch 252.0 27.7° 522.0 5.5086 197.4 148.3 

To Bench 297.0 28.5° 519.0 5.5261 168.0 125.2 

To Proof 312.0 

To Oven 359.0 36.0° 5.5476 116.8 84.6 

Out Oven 377.0 (226.5°) 187.6 140.5 



TABLE XX. 

Weights of Reducing Sugars Calculated as Milligrams of Maltose in 
Ten Grams of Dough at Different Stages of Fermentation. 

Straight Dough Straight Dough Xo Sugar Straight Dough 

2.57c. Sucrose No Sugar 2% Malt Flour 1.83% Maltose 



Sampled 
At: 


I. 

With 
Yeast 


11. 
Without 

Yeast 


III. 
With 
Yea;,t 


IV. 
Without 

Yeast 


V. 

With 
Yeast 


VI. 

W thout 
Yea.st 


VII. 
With 
Yeast 


VIII. 

Without 

Yeast 


Mixing 


481.9 


99.9 


156.0 


125.7 


231.6 


184.6 


231.0 


200.9 


1 Hour 

First 

Punch 

Second 

Punch 

To 

Bencli 

To 

Proof 

To 


456.6 
3^5.8 
274.1 


151.0 
178.2 
240.2 


209.5 
205.5 
U8.3 


159.4 
204.1 
222.0 


307.9 
278.2 
308.6 


235.7 
286.5 
326.1 


288.7 
279.3 
261.3 


230.8 
288.9 
306.0 


222,6 


260.2 


125.2 


233.6 






254.2 


312.4 










333.8 


364.3 






Oven 
Out of 
Oven 


186.8 
209.4 


256.6 
624.1 


84.6 
140.5 


245.3 


347.8 

258.7 


365-.{) 


211.9 
272.8 


320.6 



The cc)nclusion.s to be drawn from this set of results in Table XX 
are verified by considerable additional data obtained on other flours, 
as included in the second report. Curve I, Figure 7, shows the rapid 
consumption of reducing sugar by the yeast zymase in the production 
of carbon dioxide and alcohol. The fall of reducing sugar content 
slows up about the time the dough goes to the proof-cabinet, indicat- 
ing that the increased diastasis due to increased temperature is mak- 
ing itself felt. This latter increase is shown nicely by Curve II. The 
wide difference between the reducing sugars present in the two doughs 

66 



is of significance, considering that in Dough I the sugar was added 
as non-reducing sugar (sucrose). This must, therefore, be ascribed 
in part, if not entirely, to the rapid hydrolysis of sucrose by the inver- 
tase of the yeast cells. Otherwise it would likewise appear in Curve 
II. In other words, the yeast cell appears to invert the sucrose faster 
than its zymase activity requires. This same result is observed in 
every case where this sort of determination has been made. In the 
other pairs of curves the divergence is not so great because the 
amount of disaccharides present arc much smaller. 

Curve III, Figure 8, emphasizes the fact, as indicated by Curve I, 
that the yeast's requirement of available sugars continues throughout 
fermentation, and really increases with the stimulated activity during 
the proofing of the loaf. In this curve the yeast zymase has practic- 
ally exhausted the supply, and the volume of the baked loaf suffers in 
consequence, even though the flour diastase (Curve IV) is still active. 
Curves V and VI, Figure 9, on the other hand, illustrate an entirely 
different state of affairs. With a sufficient supply of diastatic power 
the excess of reducing sugars (above the yeast requirement) instead 
of falling off after three hours of fermentation as in the two previous 
cases, is carried along in an upward curve until it is no longer needed. 
The result is an increase in loaf volume. When this added diastase 
can be supplied without the associated undesirables of coloring matter 
and excessive proteoclastic action, there should be no question as to 
its importance in the manufacture of good bread. Curves VII and 
VIII, Figure 10, show much the same situation as those for I and II. 
The maltose, as sugar added at mixing, does not appear to sustain the 
fermentation in its later stages any better than sucrose. The fermen- 
tation curve, so-called, does appear to be more uniform, but whether 
that difference depends upon the nature of the sugars is still to be 
determined. 

One more point remains to be noted. The sudden increase of re- 
ducing sugars during the first few minutes in the oven is shown by 
the dotted lines at the end of curves I, II and VII. The sudden in- 
crease of heat in the oven, decreasing in rate from the exterior to the 
interior of the loaf, during what the baker terms the "oven spring," 
should be expected to steadily raise the diastase up to and beyond its 
optimum temperature. The resulting sugars produced in the loaf 
must then be taken into consideration in the interpretation of carbohy- 
drate anaylsis of bread. 



67 




30 60 90 

Time - mi/wtes 



Figure 7. 

Reducing Sugars Found in Doughs During Fermentation. 
Straight Dough. Standard Formula. Curve II. 
Straight Dough. Yeast Omitted. 



Curve I. 




minutes. 



Figure 8. 
Reducing Sugars Found in Doughs During Fermentation. 
III. Straight Dough. Sugar Omitted. Curve IV. 
Straight Dough. Yeast and Sugar Omitted. 

68 



Curve 



1 \ r 



1 — 1 — r 




r 



J \ L 



60 90 

Jime - minutes. 



J I I 



J I L. 



Figure 9. 

Reducing Sugars Found in Doughs During Fermentation. Curve V. 

Straight Dough. Diastatic Malt Added. Curve VI. 

Straight Dough With Added Diastase, 

But Yeast Omitted. 




30 CO , 

lime - minutes 

Figure 10. 

Reducing Sugars Found in Doughs During Fermentation. Curve VII. 

Straight Dough. Maltose Substituted for Sucrose. 

Curve VIII. Straight Dough. Maltose 

Substituted, Yeast Omitted. 

' 69 



The Relation of Diastatic Power to Different Forms of Starch. Be- 
fore the various diastatic preparations, such as barley malt flours and 
malt extracts, can be used intelligently in panary fermentation to sup- 
ply the required diastatic power, it is necessary to have some ra- 
tional basis for measurement. The diastatic power of these products 
should be expressed in terms of their ability to produce maltose per 
unit weight used, under the conditions of baking practice. It be- 
comes necessary first to find an easily standardized substrate which 
will be comparable to the flour which goes into the dough. The more 
accurate way of determining the diastatic activity of a malt product 
upon the particular flour with which it' is to be used offers many ob- 
jections, both technically and as a basis for control and commerce. 
The use of wheat flour as substrate involves the determination of 
the diastatic power of the flour itself, the condition or resistance of the 
starch granules to the enzyme, the acidity and bufl-"er value of the 
flour ; in short, as many factors as there are flours. 

The next possibility is in the selection of a standard starch as sub- 
strate, under controlled conditions of acidity and salt concentration, 
which will give results comparable to average bake shop conditions. 
Preliminary experiments have furnished some data which are given 
here because of their general bearing on this phase of the problem. 

Five forms of starch were used in this work. Starch sample num- 
ber 1, was the starch as it naturally occurs in flour sample No. 1009, 
with its associated diastase, and other enzymes, salts, and organic ma- 
terials. Num])"cr 2 was a sample of Merck's "soluble starch according 
to Lintner," used in water suspension without heating. No. 3 was 
the same soluble starch gelatinized by boiling. Sample No. 4 was 
commercially prepared wheat starch. Sample No. 5 was a sample 
of wheat starch prepared in this laboratory as previously described. 

The researches of Thatcher and Koch (1914), Thatcher and Ken- 
nedy (1920), and Sherman, et al. (1913-1921), furnish examples of 
the use of soluble starch as substrate for diastatic enzymes. The con- 
tribution of Swanson and Calvin (1913), showed the effect of autolstic 
diastasis on natural wheat starch. The differences between these two 
forms of substrate for diastatic activity are obvious from the pub- 
lished data. To determine whether the diastase in wheat flour would 
act more readily on added soluble starch than on the unchanged starch 
granules of the wheat itself, the following experiment was carried out. 

One 10 gram sample of flour No. 1009 was digested with 100 cc of 
water for one hour at 27° C in the regular manner. To a second 
sample were added 10 grams of soluble starch (No. 2), and handled 
in the same manner. Blanks were run for both samples to determine 
their natural reducing values. The results are given in Table XXI, 

70 



and are the averages of three or more determinations on each sample. 
There is a considerable augmentation of the maltose produced by the 
addition of a more easily digested form of starch to the diastatic 
medium. The slight increase in acidity due to the addition of the 
more acid soluble starch might account for a part of the difference, as 
shown bv the data in Table XXII. 



TABLE XXI. 

The Activity of Wheat Flour Diastase on Wheat Flour Starch 

and on added Soluble Starch. 

Difference Cal 

Actual Weight of dilated as Mai 

CU2O per 50. cc tose by Dias- 

aliquot of tase in 10. 

Solution g. Flour 

Starch Used Millif2:rams Milligrams 

10. g. Starch No. 1 (Wh't flour No. 1009 only) 82.31 2O8 ? 
10. g. Starch No. 1 (Blank) 14.1 



10. g. Starch No. 1 + 10. g. Starch No. 2 114.9 

10. g. Starch No. 1 + 10. g. Starch No. 2 (Blank) 25.6 



275.0 



The efTect of several different forms of starch on the activity of a 
diastatic malt extract was next tried. There was at hand for this 
work a fresh sample of a commercial diastatic malt syrup which had 
just been analyzed for total solids, ash, and proteins, total reducing 
sugars, and the Lintner value of which was determined as 44.4. 

The starches used as substrate were weighed out in 10 gram samples 
into 300 cc erlenmeyer flasks and shaken up with 100 cc of w^ater. To 
these there were added 10 cc of a freshly prepared 5% solution of the 
malt extract. These were allowed to digest one hour at 27° C, and 
then treated in the regular manner. Because of the high acidities of 
some of the digestion samples they were repeated, using a solution 
of K2HPO4 and KH,PO^ as buffer salts to control the pH. For con- 
venience the two sets of experiments are grouped together in table 
XXII to show^ the amounts of maltose produced by the same amounts 
of diastase under different conditions. 

An inspection of the ])H values in Table XXII, considered in rela- 
tion to the graph in Figure 3, shows that the points obtained lie for 
the most part on either side of the optimum range, and a slight change 
toward neutrality should result in a considerable increase of maltose 
[)roduction. These values for the pH are likewise rather far from 
those which normally obtain in a dough, with the exception of Starch 
N^o. 2. Consequently the maltose produced by the diastase under 
these conditions cannot be taken as a comparative measurement of 
diastatic powier of the malt extract in a dough. 

71 



TABLE XXII. 

Maltose produced by the same weight of Malt Diastase acting 

on different Starches for one hour at 27 °C. 



Starch used as Substrate 
No. 1. (Flour 1009) Sample 
No. 1. (Flour 1009) Blank 
No. 1. (Flour 1009 without added malt) 

(Sample) 

No. 1. (Flour 1009 without added malt) 

(Blank) 



pH 
at end of 
Digestion 



5.801 



Weight of CuaO Maltose Pro- 



per 50 cc of 
Digestion 
Solution 

Grams 

.25221 
.1284/ 

.0823* 

.0141* 



duced by 
Diastase of 
1 gram Malt 

Extract 
Milligrams 

247.0 



No. 2. (Soluble Starch) Sample 5.204 .1584\ 

No. 2. (Soluble Starch) Blank .1272/ 
No. 2. (Soluble Starch + Buffers) 

Sample 6.847 .2183\ 

No. 2. (Soluble Starch + Buflfers) .1772/ 

Blank 



200.8 
561.4 



No. 3. (Gelatinized Starch) Sample 
No. 3. (Gelatinized Starch) Blank 



.4119x2 
.0652x2 



2759.4 



No. 4. (Com'l Wheat Starch) Sample 4.108 

No. 4. (Com'l Wheat Starch) Blank 1.014 

No. 4. (Commercial Wheat Starch + 

Buffers) Sample 6.707 

No. 4. (Commercial Wheat Starch + 

Buffers) Blank 



.1492\ 
.1218/ 



.1458 



157.8 



136.0 



No. 5 /Prepared Wheat St'ch) Sample 4.924 
No. 5. (Prepared Wheat Starch) Blank 
No. 5. (Prepared Wheat Starch + 

Buffers) Sample 7.098 

No. 5. (Prepared Wheat Starch + 

Buffers) Blank 

*( Values taken from Table XXJ to furnish 
activity of the flour No. 1009.) 



.1470/ 
.1105 J 

.1324\ 
.1105/ 



215.2 



116.8 



a correction for the diastatic 



On the other hand, the similarity in result between Starch No. 1 
with a pH of 5.8 or less, Starch No. 2 with a pH of 5.2, and of Starch 
No. 5 with a pH at 4.9, would indicate the probable satisfactory solu- 
tion of the problem. The results for Starch sample No. 3 confirm the 
statements as reviewed earlier in this report. There can then be no 
question as to the fallacy of reporting the diastatic power of a malt 
product for bread making in terms of its action on gelatinized soluble 
starch. It is hoped that a completed report can be made on this phase 
of the work at an earlv date. 



72 



Additional analytical data on each of the fourteen flour samples 
used in these experiments is compiled for reference, and is appended 
as Table XXIII. 

TABLE XXIII. 







Analytical Data on Flour Samples. 


















Acidity 


















As Lac- 


















tic 




Baking 


ample 


Moisture 


Ash 


Protein 


Wet 


Dry 


Acid, 


pH 


Value 


No. 


% 


% 


% 


% 


% 


% 






1009 


11.61 


.423 


13.81 


31.21 


11.18 


.20 


5.981 


100 


1001 


12.15 


.405 


11.34 


35.21 


11.09 


.14 


5.816 


99 


1008 


11.70 


.459 


15.32 


44.65 


15.44 


.20 


6.133 


97 


1002 


12.14 


.610 


13.00 


41.97 


13.28 


.16 


6.052 


95 


1012 


12.72 


.407 


12.76 


38.42 


12.94 




5.732 


91 


1006 


11.96 


.378 


11.96 


35.49 


12.96 


.135 


5.777 


91 


1005 


11.27 


.431 


11.04 


29.15 


11.15 


.14 


5.843 


90 


1010 


12.48 


.539 


10.36 


30.77 


10.99 


.18 


5.961 


83 


1011 


11.43 


.562 


10.77 


29.63 


10.71 


.22 


6.05 


76 


1003 


13.06 


.463 


8.83 


25.13 


9.22 


.11 


6.044 


63 


1013 


12.83 


.543 


14.83 


44.55 


15.63 




5.596 


56 


1007 


11.06 


.637 


14.12 


36.58 


14.18 


.20 


6.132 


46 


1004 


10.58 


.829 


12.70 


29.58 


12.11 


.26 


6.166 


35 


1014 


12.22 


.795 


14.37 


39.96 


14.47 




5.843 


32 



SUMMARY 

Diastatic enzymes are recognized as one of the important factors 
which go to make up "flour strength." 

Experiments to "define more clearly the action of diastase in the 
production of better bread are indicated by a review of the literature 
dealing with the different factors of flour strength. 

The effects of diastatic enzymes of the wheat flour in panary fer- 
mentation, with respect to concentration, time, temperature, acidity, 
and diastatic power, have been determined. 

Representative flour samples, numbering fourteen in all, were col- 
lected from typical wheat growing regions of North America to serve 
as a basis for the study of diastatic enzymes in bread making. 

The baking values of the flour samples were determined by means 
of comparative baking tests. Other available analytical data has 
been tabulated along with these baking values in order to correlate 
as many different factors as possible with the "strength" of each fl-our. 

73 



Protein precipitation from cereal extracts by the tungstic acid 
reagent of Folin and Wu was studied in its application to the deter- 
mination of diastatic power in flour. A convenient, rapid, and com- 
plete removal of proteins from solution, without filtration, is effected 
by the use of 3. cc of 15% sodium tungstate per 10 grams of flour, 
acidifying with sulfuric acid to a pH of 2, with subsequent centrifug- 
ing. Diastatic activity is completely inhibited by this treatment. 

Diastatic powers of fourteen flour samples were determined bv on" 
hour autolytic digestion at 27°C. The maltose produced serves as 
a measure of the diastatic activity of that flour when in the form of 
a dough. 

^^ 

Maximum activity of flour diastase in a dough at any given tem- 
perature or pH is not reached at once because of insufficient w'ater. 
The slower hydration of the colloidal materials compensates in some 
measure for the more rapid loss of activity which the diastase would 
suffer in water suspension, and hence the rate of maltose production 
ill the dough is quite regular at any given temperature. 

Optimum hydrogen ion concentration for flour diastase, pH of 4.7, 
is seldom reached during the fermentation of a normal dough. The 
increase of acidity during fermentation, in the range of pH 6 to 5, has 
the effect of considerably increasing the production of maltose toward 
the latter part of fermentation. The effect of hydrogen ion concen- 
tration on the activity of flour diastase is in agreement with that 
found by Sherman and others for the diastase of malt. 

Temperature is the most important factor in the control of diastatic 
action in the dough. The increased temperature at proofing, along 
with the increased hydrogen ion concentration, combine to make the 
eff"ect of diastase most significant during the later stages of fermenta- 
tion. It is during this later period that the diastase produces the 
necessary sugars from which the yeast may complete the aeration 
of the dough during the proofing of the loaves. 

The flour showing the greater diastatic power should show the 
greater strength and consequently the greater baking value, providing 
the relative quality and quantity of the gluten is the same. 

A rational standard method is needed for measuring the diastatic 
materials such as are in general use in bread making. Experiments 
arc described which demonstrate the necessity for using a standard 
starch substrate, and a possible method for preparing such a sub- 
strate is suggested. 

74 



BIBLIOGRAPHY 

Alway, F. J., and Hartzell, S. 

1909. On the Strength of Wheat Flour. 23d Annual Report, 

Nebraska Agr. Exp. Sta. p. 100. 
Am. Inst. Baking. 

1921. Malt Extracts and Shortening Agents Registered by the 
American Institute of Baking. Bulletin 6. 
/Vrmstrong, IL. F. 

1910. The Chemical Properties of W'heaten Flour. Supp. to J. 

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1(^ 



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84 



i 



BIOGRAPHICAL. 

Louys A. Runisey was born May 7th, 1889, at Stryker, Ohio. He 
attended the public schools and graduated from the high school there 
in 1908. Entering Denison University, Granville, Ohio, in the fall of 
1908, he specialized in chemistry and graduated with the B. S. degree 
in June, 1912. He returned to Denison the following year and re- 
ceived the M. S. degree in June, 1913. The thesis work was done in 
the department of chemistry on some reduction compounds of tin, 
under the direction of Professor A. M. Brumbach. Following a sum- 
mer's work in the final inspection department of the Dayton Engineer- 
ing Laboratories, Dayton, Ohio, he went in October, 1913, to the 
Iowa State College of Agriculture and Mechanic Arts as an instructor 
tor in the department of chemistry. He remained there as instructor 
•in home economics and food chemistry until September, 1917, when 
he returned to Denison University as Assistant Professor of chemis- 
try. He had taken work in the graduate school of Chicago Univer- 
sity during the summer quarters of 1914 and 1916. 

After a three months' training at Fort Sheridan, Illinois, during the 
summer of 1918 he was given a certificate of Commission as second 
lieutenant and sent back to teach chemistry at Denison. 

He enrolled as a student in the graduate school. University of Min- 
nesota, in September, 1921. Having been granted a fellowship by the 
American Institute of Baking, Minneapolis, the work on his thesis was 
carried out in their laboratories, under the direction of Dr. R. A. Gort- 
ner and Dr. C. H. Bailey of the University of Minnesota, Division of 
Agricultural Biochemistry. 

In May, 1922 he presented to the faculty of the graduate school a 
thesis on "The diastatic enzymes of wheat flour and their relation to 
flour strength." in partial fulfillment of the requirements for the degree 
of Doctor of Philosophy. The degree was conferred June 14th, 1922 
by the University of Minnesota. 



85 



ACKNOV/LEDGMENT. 

The author wishes to express his gratitude and appreciation for the 
inspiration and help of Dr. Ross Aiken Gortner, under whose direction 
this investigation was carried out. Grateful acknowledgment is like- I 

wise made for the assistance and counsel of Dr. Clyde H. Bailey, and 
to the American Institute of Baking for the grant of a fellowship and 
for the facilities made available for the prosecution of this work. 

L. A. RUMSEY. 



I 



86 



